BR112020021802A2 - methods to produce a population of insulin-producing cells or a population of pancreatic beta cells and to decrease the number or inhibit the proliferation of ki67 positive cells, and, a population of pancreatic progenitor cells or cells at a later stage of differentiation - Google Patents

Abstract

METHODS FOR PRODUCING A POPULATION OF INSULIN-PRODUCING CELLS OR A BETA CELL POPULATION PANCREATIC AND TO REDUCE THE NUMBER OR INHIBIT THE PROLIFERATION OF POSITIVE CELLS FOR KI67, AND, POPULATION OF PROGENITOR CELLS PANCREATIC OR CELLS IN A LATER STAGE OF DIFFERENTIATION. The present invention relates to a method for producing a population of insulin-producing cells or a population of ß cells in the pancreas in which Ki67 positive cells are reduced, the method comprising the treatment of a population of progenitor cells endocrine cells or cells further differentiated with an inhibitor of FGFR1.

Description

1/74

METHODS TO PRODUCE A CELL POPULATION INSULIN PRODUCERS OR A CELL POPULATION

PANCREATIC BETA AND TO REDUCE THE NUMBER OR INHIBIT THE PROLIFERATION OF POSITIVE CELLS FOR KI67, E,

POPULATION OF PANCREATIC PROGENITOR CELLS OR

CELLS IN A LATER STAGE OF DIFFERENTIATION Technical Field

[001] The present invention relates to a method for removing highly proliferative Ki67 positive cells present in a population of insulin-producing cells or a population of pancreatic β cells obtained by inducing differentiation of pluripotent stem cells. [Fundamentals of Technique]

[002] Research is ongoing to induce differentiation of pluripotent stem cells, such as iPS cells or ES cells, into insulin-secreting cells, such as insulin-producing cells or pancreatic β cells, and to apply the obtained cells to the treatment of diabetes mellitus.

[003] Several approaches have been developed and reported so far, in order to induce the differentiation of pluripotent stem cells into insulin-secreting cells. Non-Patent Literature 1 states that differentiation of endocrine progenitor cells derived from human iPS cells can be promoted by treating cells with CAS192705-79-6 (1- [2-amino-6- (3,5-dimethoxyphenyl) -pyrid (2,3-d) pyrimidin-7-yl] -3-tert-butylurea), a fibroblast growth factor receptor (FGFR) 1 inhibitor.

[004] The pyrido (2,3-d) pyrimidine and naphthyridine compounds effective for inhibiting protein tyrosine kinase are known. The compounds are considered useful in the treatment of atherosclerosis, restenosis and cancer cell proliferative diseases (Patent Literature 1).

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[005] CAS192705-79-6 has been further confirmed to inhibit cell proliferation caused by stimulation of bFGF in rat myoblasts, but not to inhibit cell proliferation caused by stimulation of PDGE and to inhibit the stretching of microvessels in human placental blood vessels , suggesting its applicability as an antiproliferative agent and / or an antiangiogenic agent that targets tumor growth or the formation of new blood vessels from atherosclerotic plaques (Non-Patent Literature 2).

[006] Meanwhile, it was not previously known that a population of insulin-producing cells or a population of pancreatic β cells obtained by further inducing differentiation from a population of endocrine progenitor cells derived from pluripotent stem cells or a population of cells in a later stage of differentiation includes highly proliferative cells. In addition, no discussion has been made about the removal of such cells. Patent Literature Citation List

[007] Patent Literature 1: WO96 / 15128 Non-Patent Literature

[008] Non-Patent Literature 1: Scientific Reports 6, Article number: 35908 (2016) Non-Patent Literature 2: The journal of Pharmacology and Experimental Therapeutics, July 1998, Vol. 286 (1), p.569-577 Summary of the Invention Technical problem

[009] The inventors of the present application have found that a population of cells obtained by inducing differentiation of pluripotent stem cells into insulin-producing cells or pancreatic β cells includes highly proliferative cells distinguished for being positive for the

3/74 Ki67 marker (hereinafter, called “Ki67 positive cells”), together with these insulin-secreting cells (insulin-producing cells and pancreatic β cells).

[0010] In the case of the application of insulin-secreting cells obtained by inducing differentiation to the treatment of diabetes mellitus, etc., it is very important from the point of view of safety to strictly control cells other than insulin-secreting cells. In addition, coexisting or remaining highly proliferative cells may adversely affect receptors or influence the long-term survival of insulin-secreting cell grafts and are therefore not preferred.

Consequently, an object of the present invention is to provide an approach to remove highly proliferative Ki67 positive cells that coexist with insulin-secreting cells obtained by inducing differentiation. Solution to the Problem

[0012] The inventors of the present application conducted diligent studies to reach the object and, consequently, found that a population of endocrine progenitor cells obtained by inducing differentiation of pluripotent stem cells or a population of cells at a later stage of differentiation is treated with an FGFR1 inhibitor, in which the proliferation of Ki67 positive cells is inhibited so that a population of insulin-producing cells or a population of pancreatic β cells with a reduced Ki67 positive cell content can be obtained.

[0013] The present invention is based on these new findings and encompasses the following inventions.

[0014] [1] A method for producing an insulin-producing cell population or a pancreatic β cell population, comprising the step of treating a cell population

4/74 insulin producers or a population of pancreatic β cells with an FGFR1 inhibitor.

[0015] [2] The method according to [1], additionally comprising the stage of differentiation of the population of insulin-producing cells treated with the FGFR1 inhibitor.

[0016] [3] The method according to [1] or [2], in which the population of insulin-producing cells or the population of pancreatic β cells is treated with less than 5 µM of the FGFR1 inhibitor.

[0017] [4] The method according to any of [1] to [3], in which the cell population produced comprises cells positive for Ki67 in a proportion less than 3%.

[0018] [5] The method according to any one of [1] to [4], wherein the FGFR1 inhibitor is 1- [2-amino-6- (3,5-dimethoxyphenyl) -pyrido (2, 3- d) pyrimidin-7-yl] -3-tert-butylurea or a salt thereof.

[0019] [5-1] The method according to any one of [1] to [4], in which the FGFR1 inhibitor is a substance with a 50% inhibitory concentration (IC50) of 1 µM or less against FGFR1 .

[0020] [5-2] The method according to any one of [1] to [4], wherein the FGFR1 inhibitor is 1- [2-amino-6- (3,5-dimethoxyphenyl) -pyrido ( 2,3- d) pyrimidin-7-yl] -3-tert-butylurea (CAS No.: 192705-79-6), E-3810 (CAS No.: 1058137-23-7), PD-173074 (CAS No.: 219580-11-7), FGFR4-IN-1 (CAS No.: 1708971-72-5), FGFR-IN-1 (CAS No.: 1448169-71-8), FIIN-2 (CAS No.: 1633044-56- 0), AZD4547 (CAS No.: 1035270-39-3), FIIN-3 (CAS No.: 1637735-84-2), NVP-BGJ398 (CAS No.: 1310746-10-1), NVP-BGJ398 (CAS No.: 872511-34-7), CH5183284 (CAS No.: 1265229-25-1), Derazantinib (CAS No.: 1234356-69-4), Derazantinib Racemate, Ferulic Acid (CAS No.: 1135-24-6), SSR128129E ( CAS No.: 848318-25-2), free acid SSR128129E (CAS No.: 848463-13-8), Erdafitinib (CAS No.: 1346242-81-6), BLU9931 (CAS No.: 1538604-68-0), PRN1371 ( CAS number: 1802929-43-6), S49076 (CAS number:

5/74 1265965-22-7), LY2874455 (CAS No.: 1254473-64-7), Linsitinib (CAS No.: 867160-71-2), Dovitinib (CAS No.: 405169-16-6), Anlotinib (CAS No. : 1058156-90-3), Brivanibe (CAS No.: 649735-46-6), Derazantinib (CAS No.: 1234356-69-4), Anlotinib Dihydrochloride (CAS No.: 1360460-82-7), ACTB- 1003 ( CAS nº: 939805-30-8), BLU-554 (CAS nº: 1707289-21-1), Rogaratinibe (CAS nº: 1443530-05-9), BIBF 1120 esilate (CAS nº: 656247- 18-6), hydrochloride TG 100572 (CAS nº: 867331-64-4), ENMD-2076 (CAS nº: 934353-76-1), Alaninato de Brivanibe (CAS nº: 649735-63-7), TG 100572 (CAS nº: 867334- 05-2), BIBF 1120 (CAS No.: 656247-17-5), ENMD-2076 tartrate (CAS No.: 1291074-87-7), TSU-68 (CAS No.: 252916-29-3), Ponatinibe (No. CAS: 943319-70-8), Sulfatinib (CAS No.: 1308672-74-3), LY2784544 (CAS No.: 1229236-86-5), Dovitinib Lactate (CAS No.: 692737-80-7), SU 5402 ( CAS nº: 215543-92-3), FGF-401 (CAS nº: 1708971-55-4), tyrosine kinase- IN-1 (CAS nº: 705946-27-6), PP58 (CAS nº: 212391-58- 7), hydrochloride TG 100801 (n CAS No.: 1018069-81-2), Crenolanib (CAS No.: 670220-88-9), TG 100801 (CAS No.: 867331-82-6), Pazopanibe Hydrochloride (CAS No.: 635702- 64-6), Pazopanibe (CAS nº: 444731-52-6), PD168393 (CAS nº: 194423-15- 9), Apatinibe (CAS nº: 1218779-75-9), Palbocyclib isetionate (CAS nº: 827022-33-3), Foretinibe (CAS No.: 849217-64-7), Lenvatinibe (CAS No.: 417716-92-8), Tandutinib (CAS No.: 387867-13-2), or a salt thereof.

[0021] [6] The method according to any of [2] to [5], in which the step of differentiating the population of insulin-producing cells treated with the FGFR1 inhibitor is performed by transplantation to an animal.

[0022] [7] A method for producing a population of insulin-producing cells or a population of pancreatic β cells, comprising the step of treating a population of endocrine progenitor cells or cells in a later stage of differentiation with less than 5 µM of an FGFR1 inhibitor.

[0023] [8] A method to decrease the number or inhibit the

6/74 proliferation of Ki67 positive cells present in a population of endocrine progenitor cells or cells in a later stage of differentiation, comprising treating the cell population with an FGFR1 inhibitor.

[0024] [8-1] A method for inhibiting the proliferation of Ki67 positive cells present in a population of endocrine progenitor cells or a population of cells at a later stage of differentiation, comprising treating the cell population with an FGFR1 inhibitor .

[0025] [8-2] The method according to [8], in which the absolute number of Ki67 positive cells present in the cell population is decreased.

[0026] [8-3] The method according to any of [8], [8-1] and [8-2], in which the number of cells that are endocrine progenitor cells or cells in a later stage of differentiation and are not Ki67 positive cells present in the cell population does not decrease.

[0027] [9] The method according to any one of [8] to [8-3], in which the population of endocrine progenitor cells or cells in a later stage of differentiation is a population of cells producing insulin.

[0028] [10] The method according to [8] or [9], in which the population of endocrine progenitor cells or cells in a later stage of differentiation is treated with less than 5 µM of the FGFR1 inhibitor.

[0029] [11] The method according to any one of [8] to [10], wherein the FGFR1 inhibitor is 1- [2-amino-6- (3,5-dimethoxyphenyl) -pyrido (2, 3- d) pyrimidin-7-yl] -3-tert-butylurea or a salt thereof.

[0030] [12] A population of pancreatic progenitor cells or cells in a later stage of differentiation, comprising Ki67 positive cells in a proportion of less than 3%.

[0031] [12-1] A population of pancreatic progenitor cells or cells in a later stage of differentiation, comprising Ki67 positive cells in a proportion of less than 2%.

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[0032] [12-2] A population of pancreatic progenitor cells or cells at a later stage of differentiation, comprising Ki67 positive cells in a proportion of less than 1%.

[0033] [12A] A population of endocrine progenitor cells or cells in a later stage of differentiation, comprising Ki67 positive cells in a proportion of less than 3%.

[0034] [12A-1] A population of endocrine progenitor cells or cells in a later stage of differentiation, comprising Ki67 positive cells in a proportion of less than 2%.

[0035] [12A-2] A population of endocrine progenitor cells or cells in a later stage of differentiation, comprising Ki67 positive cells in a proportion of less than 1%.

[0036] [13] The cell population according to any of

[12] to [12-2], where the cell population is a population of insulin-producing cells.

[0037] [14] The cell population according to [12] or [13], where the cell population is used for transplantation.

[0038] [14-1] A drug that comprises a population of cells according to [12] or [13].

[0039] [14-2] A prodrug that comprises a population of cells according to [12] or [13].

[0040] [15] A method for producing an insulin-producing cell population or a pancreatic β cell population, comprising the steps of: (0) including any method described herein (for example, any method described in paragraphs [0058] a [0106]); (1) treating a population of insulin-producing cells or a population of pancreatic β cells with an FGFR1 inhibitor; and

8/74 (2) differentiation of the population of insulin-producing cells treated with the FGFR1 inhibitor.

[0041] [16] A method for producing a population of insulin-producing cells or a population of pancreatic β cells, comprising the steps of: (1) treating a population of insulin-producing cells or a population of pancreatic β cells with an FGFR1 inhibitor; and (2) incorporating the population of insulin-producing cells into a gel containing alginic acid.

[0042] [17] A method for producing an insulin-producing cell population or a pancreatic β cell population, comprising the steps of: (0) adjusting the purity of a target cell population by at least 70% or more by a method for purifying the target cell population; (1) treating a population of insulin-producing cells or a population of pancreatic β cells with an FGFR1 inhibitor; and (2) differentiation of the population of insulin-producing cells treated with the FGFR1 inhibitor.

[0043] [18] A method for the treatment of diabetes mellitus, comprising the stage of transplanting a population of cells treated with an FGFR1 inhibitor.

[0044] This specification describes the content described in the specification and / or drawings of Japanese Patent Application No. 2018- 082606 on which the priority of this application is based.

[0045] All publications, patents and patent applications cited in this document are incorporated into this document by reference in their

9/74 totality. Advantageous Effects of the Invention

[0046] The present invention can provide an approach to remove highly proliferative Ki67 positive cells that coexist with insulin secreting cells obtained by inducing differentiation. Brief Description of the Drawing

[0047] [Figure 1] Figure 1 shows the results of autopsy checks and the results of immunohistological staining on an excised graft 5 weeks after transplantation of a population of insulin-producing cells obtained by (or without) treatment with an inhibitor of FGFR1. The tip of the black arrow represents the graft. Description of Modalities

1. Terminology

[0048] In the following, the terms described here will be described.

[0049] As used herein, “about” refers to a value that can vary up to plus or minus 25%, 20%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1% of the reference value. Preferably, the term "about" or "around" refers to a range of less or more 15%, 10%, 5% or 1% of the reference value.

[0050] As used herein, "comprises (s)" or "comprising" means inclusion of the element (s) after the word, without limitation to the same (s). Consequently, it indicates the inclusion of the element (s) after the word, but it does not indicate the exclusion of any other element.

[0051] As used herein, "consists of" or "consisting of" means inclusion of all (s) the element (s) after the sentence and limitation to the same (s). Consequently, the phrase "consists of" or "consisting of" indicates that the listed element (s) is (are) necessary or essential and substantially no other element exists.

[0052] As used here, “without the use of a feeder cell (s)”

10/74 basically means that it does not contain feeder cells and does not use any preconditioned medium by culturing feeder cells. Consequently, the medium does not contain any substance, such as a growth factor or a cytokine, secreted by the feeder cells.

[0053] "Feeder cells" or "feeder" means cells that are co-cultured with another type of cells, support the cells and provide an environment that allows the cells to grow. Feeder cells can be derived from the same species or from a different species from the cells they support. For example, as a feeder for human cells, human skin fibroblasts or human embryonic stem cells can be used or a primary culture of murine embryonic fibroblasts or immortalized murine embryonic fibroblasts can be used. Feeder cells can be inactivated by exposure to radiation or treatment with mitomycin C.

[0054] As used herein, “adhered (adherent)” refers to cells that are attached to a container, for example, the cells are attached to a cell culture plate or a flask made of sterile plastic (or plastic coated) in the presence of an appropriate medium. Some cells cannot be maintained or grown in culture without adhering to the cell culture vessel. In contrast, non-adherent cells can be maintained and proliferate in culture without adhering to the container.

[0055] As used herein, "culture" refers to the maintenance, growth and / or differentiation of cells in an in vitro environment. "Culture" means to maintain, proliferate and / or differentiate cells outside tissue or the living body, for example, in a cell culture plate or flask. The culture includes two-dimensional culture (flat culture) and three-dimensional culture (suspension culture).

[0056] As used here, "enrich (s)" and "enrich" refer to increasing the amount of a given component in a

11/74 composition, such as a cell composition and "enriched" refers, when used to describe a cell composition, for example, a cell population, to a cell population increased in the amount of a given component in comparison with the percentage of such a component in the cell population before enrichment. For example, a composition such as a cell population can be enriched for a target cell type and, consequently, the percentage of the target cell type is increased compared to the percentage of target cells present in the cell population before enrichment. A cell population can be enriched for a target cell type by a cell selection and classification method known in the art. A population of cells can be enriched by a specific classification or selection process described here. In a given embodiment of the present invention, a cell population is enriched for a target cell population by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% by a method of enriching the target cell population.

[0057] As used herein, "depletes (m)" and "depletion" refer to decreasing the amount of a particular component in cells or a composition, such as a cell composition and "depleted" refers to, when used to describe cells or a composition of cells, for example, a cell population, to a cell population decreased by the amount of a given component compared to the percentage of that component in the cell population before depletion. For example, a composition such as a cell population can be depleted for a target cell type and, consequently, the percentage of the target cell type is decreased compared to the percentage of target cells present in the cell population before depletion. A cell population can be depleted for one type of cell.

12/74 target cell by a cell selection and classification method known in the art. A cell population can be depleted by a specific sorting or selection process described here. In a given embodiment of the present invention, a cell population is reduced (depleted) to a target cell population by at least 50%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% by a method of depleting a target cell population.

[0058] As used herein, "purifies (m)" and "purification" refer to removing impurities in a composition, such as a cell composition and making it pure for a particular component and "purified" refers to , when used to describe a cell composition, for example, a cell population, to a cell population in which the amount of impurities is decreased compared to the percentage of such components in the cell population before purification and the purity of a particular component is improved. For example, a composition such as a cell population can be purified for a target cell type and, consequently, the percentage of the target cell type is increased compared to the percentage of target cells present in the cell population before purification. A cell population can be purified for a target cell type by a cell selection and classification method known in the art. A population of cells can be purified by a specific sorting or selection process described here. In a certain embodiment of the present invention, the purity of a target cell population is brought about by a method of purifying a target cell population by at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or to the extent that impurities (including contaminating cells) are undetectable.

[0059] As used herein, “do not decrease the number of cells” means that the number of cells does not decrease markedly due to

13/74 execution of the method of the present invention and means that there is no marked difference between the number of cells before the execution of the method and the number of cells after the execution of the method. However, a decrease in the number of cells may occur that is not caused by the execution of the method of the present invention (for example, natural cell death that can generally occur in conventionally known cell culture and differentiation steps). Thus, "not decreasing the number of cells" also includes the case where the rate of decrease in the number of cells after the execution of the method of the present invention from that before the execution is 30% or less, 20% or less, 10% or less, or 5% or less.

[0060] As used herein, "suppress proliferation" means that the number of cells is not markedly increased due to the execution of the method of the present invention and means that there is no marked increase between the number of cells before the execution of the method and the number of cells after executing the method. "Suppressing proliferation" also includes the case where the rate of increase in the number of cells after executing the method of the present invention from that before executing is 30% or less, 20% or less, 10% or less, or 5% or less.

[0061] As used herein, "marker" means a cellular antigen or a gene thereof that is specifically expressed depending on a predetermined cell type, such as "marker protein" and "marker gene". Preferably, a marker is a cell surface marker and this allows for the concentration, isolation and / or detection of living cells. A marker can be a positive selection marker or a negative selection marker.

[0062] Detection of a marker protein can be conducted by an immunological assay, for example, ELISA, immunostaining or flow cytometry using an antibody specific for the marker protein. The detection of a marker gene can be conducted by a method of

14/74 amplification and / or detection of nucleic acid known in the art, for example, RT-PCR, microarray, biochip or the like. As used herein, "positive" for a marker protein means to be detected as positive by flow cytometry and "negative" therefore it means to be equal to or less than the lower detection limit in flow cytometry. In addition, "positive" for a marker gene means to be detected by RT-PCR and "negative" therefore it means to be equal to or less than the lower detection limit in RT-PCR.

[0063] As used herein, "expression" is defined as the transcription and / or translation of a given nucleotide sequence conducted by an intracellular promoter.

[0064] As used herein, "factor with CDK8 / 19 inhibitory activity" means any substance with CDK8 / 19 inhibitory activity. CDK8, unlike other proteins in the same CDK family, is not necessary for cell proliferation. Inhibition of CDK8 has little effect under usual conditions. CDK19 and CDK8 are similar to each other, as mentioned above. Normally, inhibition of CDK8 also involves inhibition of CDK19.

[0065] "Growth factors" are endogenous proteins that promote the differentiation and / or proliferation of specific cells. Examples of “growth factors” include epidermal growth factor (EGF), acid fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), similar growth factor insulin 1 (IGF-1), insulin-like growth factor 2 (IGF-2), keratinocyte growth factor (KGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), beta-transforming growth hormone (TGF-β), vascular endothelial growth factor (VEGF), transferrin, various interleukins (eg, IL-1 to IL-18), various factors stimulating

15/74 colonies (for example, granulocyte / macrophage colony stimulating factor (GM-CSF)), various interferons (for example, IFN-γ, and the like), and other cytokines with effects on stem cells, for example, stem cell factor (SCF) and erythropoietin (Epo).

[0066] As used herein, "ROCK inhibitors" means substances that inhibit Rho kinase (ROCK: associated with Rho, supercoiled helix containing protein kinase) and can be substances that inhibit ROCK I and ROCK II. ROCK inhibitors are not particularly limited, as long as they have the function mentioned above and examples include N- (4-pyridinyl) -4β - [(R) -1-aminoethyl] cyclohexane-1α-carboxamide (which can be here also called I-27632), Fasudil (HA1077), (2S) -2-methyl-1 - [(4-methyl-5-isoquinolinyl] sulfonyl] hexahydro-1H-1,4-diazepine (H- 1152), 4β- [(1R) -1-aminoethyl] -N- (4-pyridyl) benzene-1zencarboxamide (Wf-536), N- (1H-pyrrole [2,3-b] pyridin-4-yl) -4PER (R) -1-aminoethyl] cyclohexane-1α-carboxamide (Y-30141), N- (3 - {[2- (4-amino-1,2,5-oxadiazol-3-yl) - 1-ethyl-1H-imidazo [4,5-c] pyridin-6-yl] oxy} phenyl) -4 - {[2- (4-morpholinyl) ethyl] -oxy} benzamide (GSK269962A), N- (6 -fluoro-1H-indazol-5-yl) -6-methyl-2-oxo-4- [4- (trifluoromethyl) phenyl] -3,4-dihydro-1H-pyridine-5-carboxamide (GSK429286A). ROCK inhibitors are not limited to these and antisense oligonucleotides and siRNA for ROCK mRNA, antibodies that bind to ROCK and dominant negative ROCK mutants can also be used, commercially available or synthesized according to a method known as ROCK inhibitors.

[0067] As used herein, "GSK3β inhibitors" are substances with GSK3β (glycogen synthase kinase 3β) inhibitory activity. GSK3 (glycogen synthase kinase 3) is a serine / threonine kinase protein and is involved in many signaling pathways associated with glycogen production, apoptosis, stem cell maintenance, etc. GSK3 has the 2 isoforms α and β. "GSK3β inhibitors" used in the present invention are not particularly

16/74 limited, as long as they have GSK3β inhibitory activity and can be substances with GSK3α inhibitory activity and GSK3β inhibitory activity.

[0068] Examples of GSK3β inhibitors include CHIR98014 (2 - [[2- [(5-nitro-6-aminopyridin-2-yl) amino] ethyl] amino] -4- (2,4-dichlorophenyl) -5- ( 1H- imidazol-1-yl) pyrimidine), CIR99021 (6 - [[2 - [[4- (2,4-dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) -2-pyrimidinyl ] amino] ethyl] amino] nicotinonitrile), TDZD-8 (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione), SB216763 (3- (2,4-dichlorophenyl) -4 - (1-methyl-1H-indol-3-yl) -1H-pyrrol-2,5-dione), TWS-119 (3- [6- (3-aminophenyl) -7H-pyrrole [2,3-d ] pyrimidin-4-yloxy] phenol), kenpaulone, 1-azakenpaulone, SB216763 (3- (2,4-dichlorophenyl) -4- (1-methyl-1H-indol-3-yl) -1H-pyrrole-2, 5-dione), SB415286 (3 - [(3-chloro-4-hydroxyphenyl) amino] -4- (2-nitrophenyl) -1H-pyrrole-2,5-dione), and AR-AO144-18, CT99021, CT20026, BIO, BIO-acetoxime, cyclopentadienyl pyridocarbazole-ruthenium complex, OTDZT, alpha-4-dibromoacetophenone, lithium, and the like. GSK3β is not limited to these and antisense oligonucleotides and siRNA for GSK3β mRNA, antibodies that bind to GSK3β, dominant negative GSK3β mutants, and the like can also be used, commercially available, or synthesized according to a method known as inhibitors of GSK3β.

[0069] As used herein, examples of “serum replacement” include serum replacement Knockout (KSR: Invitrogen), serum replacement StemSure (Wako), B-27 supplement, N2 supplement, albumin (eg lipid-rich albumin), insulin, transferrin, fatty acids, collagen precursors, trace elements (eg zinc, selenium (eg sodium selenite)), 2-mercaptoethanol, 3'-thiolglycerol or mixtures thereof (eg example, ITS-G). Preferred serum substitutions are B-27 supplement, KSR, StemSure serum substitution, ITS-G. The serum replacement concentration in a medium when

17/74 added to a medium is 0.01 to 10% by weight and, preferably, 0.1 to 2% by weight. In the present invention, "serum replacement" is preferably used instead of serum.

2. Population of endocrine progenitor cells or cell population at a later stage of differentiation with suppressed proliferation of Ki67 positive cells

[0070] The present invention relates to a population of endocrine progenitor cells or a population of cells in a later stage of differentiation with suppressed proliferation of Ki67 positive cells. These cell populations can be obtained by treatment with an FGFR1 inhibitor.

[0071] As used herein, "Ki67 positive cells" means cells that coexist with endocrine progenitor cells or cells in a later stage of differentiation at least in a population of endocrine progenitor cells or a population of cells in a later stage of differentiation obtained by inducing differentiation from pluripotent stem cells in the process of differentiating pluripotent stem cells from pancreatic β cells, and are distinguished by the expression of Ki67 as a marker.

[0072] "Ki67" is known as a cell cycle-related nucleoprotein and is also known as a marker for cell proliferation and the cell cycle because its expression is verified in phases G1, S, G2 and M of proliferating cells and not it is verified in Phase G0, a quiescent stage.

[0073] It is known that cells with different characteristics depending on the differentiation stages appear in the process of differentiating pluripotent stem cells into pancreatic β cells (WO2009 / 012428 and WO2016 / 021734). For example, differentiation stages can be broadly classified into pluripotent stem cells,

18/74 definitive endodermal cells, primitive digestive tract cells, posterior cells of the anterior intestine, pancreatic progenitor cells, endocrine progenitor cells, insulin-producing cells and pancreatic β cells in order of ways relatively undifferentiated to differentiated forms.

[0074] As used here, “pluripotency” means the ability to differentiate into tissues and cells with various different shapes and functions and to differentiate into cells of any lineage of the 3 germ layers. “Pluripotency” is different from “totipotence”, which is the ability to differentiate into any tissue in the living body, including the placenta, in which pluripotent cells cannot differentiate in the placenta and therefore do not have the ability to form a individual.

[0075] As used here, "multipotency" means the ability to differentiate into plural and limited numbers of cell lines. For example, mesenchymal stem cells, hematopoietic stem cells and neural stem cells are multipotent, but not pluripotent.

[0076] As used herein, “pluripotent stem cells” refers to embryonic stem cells (ES cells) and cells potentially with a pluripotency similar to that of ES cells, that is, the ability to differentiate into various tissues (all endodermic, mesodermal, and ectodermal tissues) in the living body. Examples of cells with a pluripotency similar to that of ES cells include "induced pluripotent stem cells" (which can also be called "iPS cells" here). In the present invention, preferably, pluripotent stem cells are human pluripotent stem cells.

[0077] Available "ES cells" include murine ES cells, such as several murine ES cell lines established by inGenious, RIKEN and the like, and human ES cells, such as several human ES cell lines established by NIH, RIKEN, University of Kyoto,

19/74 Cellartis, and the like. For example, available ES cell lines include CHB-1 to CHB-12, RUES1, RUES2, HUES1 to HUES28 from NIH and the like; H1 and H9 from WisCell Research; and KhES-1, KhES-2, KhES-3, KhES-4, KhES-5, SSES1, SSES2, SSES3 by RIKEN and the like.

[0078] "Induced pluripotent stem cells" refers to cells that are obtained by reprogramming somatic mammalian cells or undifferentiated stem cells through the introduction of particular factors (nuclear reprogramming factors). Currently, there are several “induced pluripotent stem cells” and iPS cells established by Yamanaka, et al. introducing the 4 factors Oct3 / 4, Sox2, Klf4, c-Myc into murine fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676); iPS cells derived from human cells, established by the introduction of 4 similar factors in human fibroblasts (Takahashi K, Yamanaka S., et al. Cell, (2007) 131: 861-872.); Nanog-iPS cells established by cell classification using Nanog expression as an indicator after the introduction of the 4 factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317. ); iPS cells produced by a method that does not use c-Myc (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106); and iPS cells established by the introduction of 6 factors in a virus-free form (Okita K et al. Nat. Methods 2011 May; 8 (5): 409-12, Okita K et al. Stem Cells. 31 (3) 458- 66) can also be used. In addition, induced pluripotent stem cells established by the introduction of 4 factors OCT3 / 4, SOX2, NANOG and LIN28 by Thomson et al. (Yu J., Thomson JA. Et al., Science (2007) 318: 1917-1920.); induced pluripotent stem cells produced by Daley et al. (Park IH, Daley GQ.et al., Nature (2007) 451: 141-146); induced pluripotent stem cells produced by Sakurada et al. (Unexamined Japanese Patent Application Publication No. 2008-307007) and the like can be used.

[0079] In addition, any of the pluripotent stem cells

20/74 induced reactions known in the art described in all published articles (for example, Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol 3, Issue 5, 568- 574; Kim JB., Scholer HR., Et al., Nature, (2008) 454, 646-650; Huangfu D., Melton, DA., Et al., Nature Biotechnology, (2008) 26, No. 7, 795-797) or patents (for example, Unexamined Japanese Patent Application Publication No. 2008-307007, Unexamined Japanese Patent Application Publication No. 2008-283972, US2008-2336610, US2009-047263, WO2007- 069666, WO2008-118220, WO2008-124133, WO2008-151058, WO2009- 006930, WO2009-006997, WO2009-007852) can be used.

[0080] The induced pluripotent cell lines available include several iPS cell lines established by NIH, Institute of Physical and Chemical Research (RIKEN), Kyoto University and the like. For example, these human iPS cell lines include the RIKEN HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A and Nips-B2 cell lines and the Kyoto University cell lines, Ff-WJ-18, Ff - I01s01, Ff-I01s02, Ff-I01s04, Ff-I01s06, Ff-I14s03, Ff-I14s04, QHJI01s01, QHJI01s04, QHJI14s03, QHJI14s04, 253G1, 201B7, 409A iPS MyCell (21525.102.10A), iPS MyCell cells (21526.101.10A), and the like.

[0081] As used herein, "pancreatic progenitor cell population" means a population of cells distinguished by pancreatic progenitor cells. As used herein, pancreatic progenitor cells mean cells distinguished by the expression of at least one of the PDX-1, NKX6-1, PTF-1α, GATA4 and SOX9 markers.

[0082] The pancreatic progenitor cell population is a cell population comprising pancreatic progenitor cells in a proportion of 30% or more, preferably 50% or more, more preferably 70% or more. The population of pancreatic progenitor cells may include other cells (for example, progenitor cells

21/74 endocrines, insulin-producing cells and Ki67 positive cells), in addition to pancreatic progenitor cells.

[0083] As used herein, "endocrine progenitor cell population" means a population of cells distinguished by endocrine progenitor cells.

[0084] As used herein, endocrine progenitor cells mean cells distinguished by the expression of at least one of the markers Chromogranin A, NeuroD and NGN3 and no expression of a marker of the pancreas-related hormonal system (for example, insulin). Endocrine progenitor cells can express a marker, such as PAX-4, NKX2-2, Islet-1, PDX-1 or PTF-1α.

The population of endocrine progenitor cells is a population of cells comprising endocrine progenitor cells in a proportion of 30% or more, preferably 50% or more, more preferably 70% or more. The population of endocrine progenitor cells may include other cells (for example, pancreatic progenitor cells, insulin-producing cells and Ki67 positive cells), in addition to endocrine progenitor cells.

[0086] The proportion of specific cells in a cell population can be determined based on a known approach, capable of calculating the number of cells, such as flow cytometry.

[0087] "Cell population at a later stage of differentiation" means a population of cells distinguished by cells whose differentiation stage in pancreatic β cells is more advanced than that of endocrine progenitor cells. Cells whose stage of differentiation into pancreatic β cells is more advanced than endocrine progenitor cells are preferably insulin-producing cells or pancreatic β cells.

[0088] As used herein, “insulin-producing cells” means

22/74 cells distinguished by the fact that the expression of an insulin marker is verified and the level of expression of NGN3 is in a proportion of less than 1/3 of the maximum expression confirmed in endocrine progenitor cells. "Insulin-producing cells" are cells that can express an NKX6.1 marker and preferably express both insulin and NKX6.1 markers and have an NGN3 expression level in a proportion of less than 1/3 of the maximum confirmed expression in endocrine progenitor cells.

[0089] The level of expression of NGN3 can be measured by quantitative RT-PCR. The cDNA prepared from cells at a predetermined stage using the Cells-to-CT kit (Thermo Fisher Scientific) is used in the measurement using sets of TaqMan probe / primer sequences against NGN3 and GAPDH and then an expression level relative to the level of expression of GAPDH is considered as the level of gene expression of NGN3.

[0090] The "population of insulin-producing cells" is a population of cells comprising insulin-producing cells in a proportion of 5% or more, preferably 15% or more, more preferably 30% or more. The cell population may include other cells (for example, endocrine progenitor cells; other pancreatic hormone-producing cells that express at least one of the glucagon, somatostatin, and pancreatic polypeptide markers; and Ki67-positive cells), in addition to the insulin-producing cells.

[0091] The population of insulin-producing cells can be divided into “population of early insulin-producing cells” that appears in an early stage of a process of differentiation of endocrine progenitor cells and “population of late-insulin-producing cells” that appears at a more advanced stage of differentiation. The population of cells producing late insulin can be distinguished,

23/74 particularly, by the inclusion of NKX6.1 positive / insulin positive cells (INS / NKX6.1 double positive cells) in a proportion of 20% or more.

[0092] As used herein, "pancreatic β cells" means cells that are more mature than "insulin-producing cells" and, specifically, means cells distinguished for expressing at least one of the MAFA, UCN3 and IAPP markers, which are maturation markers of pancreatic β cells, or by a reaction to increase insulin secretion by stimulating glucose.

[0093] "Pancreatic β cell population" is a cell population comprising pancreatic β cells that can be obtained by differentiation and / or maturation, preferably differentiation and / or maturation in vivo, of a population of endocrine progenitor cells or a cell population at a later stage of differentiation. The cell population may include other cells (for example, insulin-producing cells and Ki67 positive cells), in addition to pancreatic β cells.

[0094] The population of endocrine progenitor cells or the population of cells at a later stage of differentiation can be obtained by using a known approach to induce differentiation of pluripotent stem cells into pancreatic β cells. Specifically, each cell population of interest can be obtained using the following differentiation induction steps: step 1) induce the differentiation of pluripotent stem cells into definitive endodermal cells; step 2) induce the differentiation of definitive endodermal cells into primitive cells of the digestive tract; step 3) induce the differentiation of the primitive cells of the digestive tract into posterior cells of the anterior intestine; step 4) induce differentiation of the posterior cells of the

24/74 anterior intestine in pancreatic progenitor cells; step 5) induce the differentiation of pancreatic progenitor cells into endocrine progenitor cells; and step 6) induce the differentiation of endocrine progenitor cells into insulin-producing cells.

[0095] In the following, each step will be described, although the induction of differentiation in each cell is not limited by these approaches. Step 1) Differentiation in definitive endodermal cells

[0096] Pluripotent stem cells are first allowed to differentiate into permanent endodermal cells. Methods for inducing the definitive endoderm from pluripotent stem cells are already known, and either method can be used. Preferably, pluripotent stem cells are cultured in a medium containing activin A, more preferably a medium containing activin A, a ROCK inhibitor and a GSK3β inhibitor, to differentiate into definitive endodermal cells. The number of cells at the beginning of the culture is not particularly limited and is 22,000 to 150,000 cells / cm2, preferably 22,000 to

100,000 cells / cm2, more preferably from 22,000 to 80,000 cells / cm2. The culture period is 1 day to 4 days, preferably 1 day to 3 days, particularly preferably 3 days.

[0097] The temperature of the culture is not particularly limited and the culture is carried out at 30 to 40 ° C (for example, 37 ° C). The concentration of carbon dioxide in a culture vessel is on the order of, for example, 5%.

[0098] The medium used in this step can be a basal medium for use in mammalian cell culture, such as RPMI 1640 medium, MEM medium, iMEM medium, DMEM / F12 medium, improved MEM zinc option medium, improved MEM medium / 1% B27 / penicillin-streptomycin or MCDB131 medium / 20 mM Glucose / NaHCO3 / FAF-BSA / ITS-X / Glutamax / acid

25/74 ascorbic / penicillin-streptomycin.

The concentration of activin A in the medium is generally 30 to 200 ng / ml, preferably 50 to 150 ng / ml, more preferably 70 to 120 ng / ml, particularly preferably about 100 ng / ml.

[00100] In another embodiment, activin A may be contained in a low dose, for example, in an amount of 5 to 100 ng / ml, preferably 5 to 50 ng / ml, more preferably 5 to 10 ng / ml, in the quite.

[00101] In an alternative embodiment, the concentration of activin A in the medium is about 0.1 to 100 ng / ml, preferably about 1 to 50 ng / ml, more preferably about 3 to 10 ng / ml.

[00102] The concentration of the GSK3β inhibitor in the medium is appropriately defined depending on the type of GSK3β inhibitor used. For example, in the case of using CHIR99021 as the GSK3β inhibitor, its concentration is generally 2 to 5 µM, preferably 2 to 4 µM, particularly preferably about 3 µM.

[00103] The concentration of the ROCK inhibitor in the medium is appropriately defined depending on the type of ROCK inhibitor used. For example, in the case of using Y27632 as the ROCK inhibitor, its concentration is generally 5 to 20 µM, preferably 5 to 15 µM, particularly preferably about 10 µM.

[00104] The medium can be additionally supplemented with insulin. Insulin can be contained in an amount of 0.01 to 20 µM, preferably 0.1 to 10 µM, more preferably 0.5 to 5 µM, in the medium. The concentration of insulin in the medium may be, but is not limited to, the concentration of insulin contained in the added B-27 supplement.

[00105] Specifically, cells are cultured for 1 day in a medium containing activin A, a ROCK inhibitor and a GSK3β inhibitor and then cultured for 2 days in a medium containing only activin A with the medium replaced by a new every day. Alternatively, stem cells

26/74 pluripotent trunks can be subjected to the first culture in a medium containing 0.01 to 20 µM of insulin in the presence of a low dose of activin A and subsequently subjected to a second culture in an insulin-free medium, for production. Step 2) Differentiation in primitive cells of the digestive tract

[00106] The definitive endodermal cells obtained in step 1) are additionally cultured in a medium containing a growth factor to induce their differentiation in primitive cells of the digestive tract. The culture period is from 2 days to 8 days, preferably about 4 days.

[00107] The temperature of the culture is not particularly limited and the culture is carried out from 30 to 40 ° C (for example, 37 ° C). The concentration of carbon dioxide in a culture vessel is on the order of, for example, 5%. The culture can be performed by any two-dimensional culture and three-dimensional culture.

[00108] A basal medium for use in culturing mammalian cells can be used as a culture medium, as in step 1). The medium can be adequately supplemented with a replacement of serum, a vitamin, an antibiotic and the like, in addition to the growth factor.

The growth factor is preferably EGF, KGF and / or FGF10, more preferably EGF and / or KGF, even more preferably KGF.

[00110] The concentration of growth factor in the medium is appropriately defined depending on the type of growth factor used and is generally about 0.1 nM to 1000 µM, preferably about 0.1 nM to 100 µM. In the case of EGF, its concentration is about 5 to 2000 ng / ml (ie, about 0.8 to 320 nM), preferably about 5 to 1000 ng / ml (ie, about 0.8 to 160 nM), more preferably about 10 to 1000 ng / ml (i.e., about 1.6 to 160 nM). In the case of FGF10, its concentration is about 5 to 2000 ng / ml (that is, about 0.3 to 116 nM), preferably

27/74 about 10 to 1000 ng / ml (i.e., about 0.6 to 58 nM), more preferably about 10 to 1000 ng / ml (i.e., about 0.6 to 58 nM). For example, in the case of using KGF as a growth factor, its concentration is generally 5 to 150 ng / ml, preferably 30 to 100 ng / ml, particularly preferably about 50 ng / ml. Step 3) Differentiation in posterior cells of the anterior intestine

[00111] The primitive cells of the digestive tract obtained in step 2) are additionally cultured in a medium containing a growth factor, cyclopamine, noggin and the like to induce their differentiation in posterior cells of the anterior intestine. The culture period is from 1 day to 5 days, preferably about 2 days. The culture can be performed by any two-dimensional culture and three-dimensional culture.

[00112] The temperature of the culture is not particularly limited and the culture is carried out at 30 to 40 ° C (for example, 37 ° C). The concentration of carbon dioxide in a culture vessel is on the order of, for example, 5%.

[00113] As in step 1), a basal medium for use in culturing mammalian cells can be used as a culture medium. The medium can be adequately supplemented with a replacement of serum, a vitamin, an antibiotic and the like, in addition to the growth factor.

The growth factor is preferably EGF, KGF and / or FGF10, more preferably EGF and / or KGF, even more preferably KGF.

[00115] The concentration of growth factor in the medium is appropriately defined depending on the type of growth factor used and is generally about 0.1 nM to 1000 µM, preferably about 0.1 nM to 100 µM. In the case of EGF, its concentration is about 5 to 2000 ng / ml (ie, about 0.8 to 320 nM), preferably about 5 to 1000 ng / ml (ie, about 0.8 to 160 nM), more preferably about 10 to 1000 ng / ml

28/74 (i.e., about 1.6 to 160 nM). In the case of FGF10, its concentration is about 5 to 2000 ng / ml (that is, about 0.3 to 116 nM), preferably about 10 to 1000 ng / ml (that is, about 0.6 to 58 nM), more preferably about 10 to 1000 ng / ml (i.e., about 0.6 to 58 nM). For example, in the case of using KGF as a growth factor, its concentration is generally 5 to 150 ng / ml, preferably 30 to 100 ng / ml, particularly preferably about 50 ng / ml.

[00116] The concentration of cyclopamine in the medium is not particularly limited and is generally 0.5 to 1.5 µM, preferably 0.3 to 1.0 µM, particularly preferably about 0.5 µM.

The concentration of noggin in the medium is not particularly limited and is generally 10 to 200 ng / ml, preferably 50 to 150 ng / ml, particularly preferably about 100 ng / ml. Step 4) Differentiation in pancreatic progenitor cells

[00118] The posterior cells of the anterior intestine obtained in step 3) are additionally cultured in a medium containing a factor with CDK8 / 19 inhibitory activity, preferably a medium containing a factor with CDK8 / 19 inhibitory activity and a growth factor, to induce their differentiation into pancreatic progenitor cells. The culture period is from 2 days to 10 days, preferably about 5 days. The culture can be performed by any two-dimensional culture and three-dimensional culture.

[00119] In the case of two-dimensional culture, according to the previous report (Toyoda et al., Stem cell Research (2015) 14, 185-197), the posterior cells of the anterior intestine obtained in step 3) are treated and dispersed by pipetting with 0.25% trypsin-EDTA and the dispersion is subjected to centrifugal separation to obtain the cell suspension, and then the suspension is seeded again in fresh medium from step 4.

[00120] As in step 1), a basal medium for use in culturing mammalian cells can be used as a culture medium. The medium can be

29/74 adequately supplemented with a replacement of serum, a vitamin, an antibiotic and the like, in addition to the growth factor.

[00121] Each of the compounds mentioned above or their salts can be used as the factor with CDK8 / 19 inhibitory activity. The amount of the factor added to the medium is determined appropriately according to the compound or the salt thereof used and is generally about 0.00001 µM to 5 µM, preferably 0.00001 µM to 1 µM. The concentration of the factor with CDK8 / 19 inhibitory activity in the medium is preferably at a concentration that reaches 50% or more inhibitory activity for CDK8 / 19.

The growth factor is preferably EGF, KGF and / or FGF10, more preferably KGF and / or EGF, even more preferably KGF and EGF.

[00123] The concentration of growth factor in the medium is appropriately defined depending on the type of growth factor used and is generally about 0.1 nM to 1000 µM, preferably about 0.1 nM to 100 µM. In the case of EGF, its concentration is about 5 to 2000 ng / ml (ie, about 0.8 to 320 nM), preferably about 5 to 1000 ng / ml (ie, about 0.8 to 160 nM), more preferably about 10 to 1000 ng / ml (i.e., about 1.6 to 160 nM). In the case of FGF10, its concentration is about 5 to 2000 ng / ml (that is, about 0.3 to 116 nM), preferably about 10 to 1000 ng / ml (that is, about 0.6 to 58 nM), more preferably about 10 to 1000 ng / ml (i.e., about 0.6 to 58 nM). For example, in the case of using KGF and EGF as a growth factor, the concentration of EGF is generally 5 to 150 ng / mL, preferably 30 to 100 ng / mL, particularly preferably about 50 ng / mL, and the concentration of KGF is generally 10 to 200 ng / ml, preferably 50 to 150 ng / ml, particularly preferably about 100 ng / ml.

[00124] The culture on the first day in step 4) can be performed on

30/74 presence of a ROCK inhibitor, and the culture in the following days can be carried out in a medium without ROCK inhibitor.

[00125] The medium can also contain a PKC activator. PdBU (PKC II activator), TPB (PKC V activator), or similar is used as the PKC activator, although the PKC activator is not limited to them. The concentration of the PKC activator to be added is about 0.1 to 100 ng / ml, preferably about 1 to 50 ng / ml, more preferably about 3 to 10 ng / ml.

[00126] The medium can also be supplemented with dimethyl sulfoxide and / or activin (1 to 50 ng / ml).

[00127] In any of the steps, the medium can be supplemented with a serum substitution (for example, supplement B-27, ITS-G), in addition to the components described above. In addition, an amino acid, L-glutamine, GlutaMAX (product name), a non-essential amino acid, a vitamin, nicotinamide, an antibiotic (for example, Antibiotic-Antimycotic (also called AA here), penicillin, streptomycin, or a mixture), an antimicrobial agent (eg amphotericin B), an antioxidant, pyruvic acid, a buffer, inorganic salts and the like can be added to them if necessary. In the case of the addition of an antibiotic to the medium, its concentration in the medium is normally 0.01 to 20% by weight, preferably 0.1 to 10% by weight. The culture can be performed by any two-dimensional culture and three-dimensional culture.

[00128] In the case of two-dimensional culture, cell culture is performed by adherent culture without the use of feeder cells. For culture, a culture vessel is used, for example, a plate, flask, microplate or cell culture sheet, such as OptiCell (product name) (Nunc). The culture vessel is preferably surface treated to improve cell adhesion (hydrophilicity), or coated with a substrate for cell adhesion, such as collagen, gelatin,

31/74 poly-L-lysine, poly-D-lysine, laminin, fibronectin, Matrigel (e.g., BD Matrigel (Nippon Becton Dickinson Company, Ltd.)) or vitronectin. The culture vessel is preferably a culture vessel coated with type I collagen, Matrigel, fibronectin, vitronectin or poly-D-lysine, more preferably a culture vessel coated with Matrigel or poly-D-lysine.

[00129] The temperature of the culture is not particularly limited and the culture is carried out at 30 to 40 ° C (for example, 37 ° C). The concentration of carbon dioxide in a culture vessel is on the order of, for example, 5%.

[00130] Pancreatic progenitor cells obtained in step 4) can be further purified using a known surface marker glycoprotein 2 (GP2) or similar. Purification can be carried out by a method known per se, for example, using granules immobilized with anti-GP2 antibody. Step 5) Differentiation in endocrine progenitor cells

[00131] The endocrine progenitor cells obtained in step 4) are additionally cultured in a medium containing a growth factor to induce their differentiation into endocrine progenitor cells. The culture can be performed by any two-dimensional culture and three-dimensional culture. In the case of two-dimensional culture, the pancreatic progenitor cells obtained in step 4) are treated and dispersed by pipetting with 0.25% trypsin-EDTA and the dispersion is subjected to centrifugal separation to obtain the cell suspension and then to suspension is sown again in fresh medium from step 5). The culture period is from 2 days to 3 days, preferably about 2 days.

[00132] As in step 1), a basal medium for use in culturing mammalian cells can be used as a culture medium. The medium is supplemented with SANT1, retinoic acid, ALK5 II, T3 and LDN inhibitor

32/74 according to the previous report (Nature Biotechnology 2014; 32: 1121-1133) and can be appropriately supplemented with a Wnt inhibitor, a ROCK inhibitor, FGF (preferably FGF2), a serum substitute, a vitamin, an antibiotic and the like.

[00133] The culture is performed by non-adherent culture without the use of feeder cells. For culture, a plate, flask, microplate, porous plate (Nunc), or similar, or a bioreactor is used. The culture vessel is preferably surface treated to decrease cell adhesion.

[00134] The temperature of the culture is not particularly limited and the culture is carried out from 30 to 40 ° C (for example, 37 ° C). The concentration of carbon dioxide in a culture vessel is on the order of, for example, 5%. Step 6) Differentiation in insulin-producing cells

[00135] The endocrine progenitor cells obtained in step 5) are additionally cultured in a medium containing a growth factor to induce their differentiation into insulin-producing cells. The culture period is 10 days to 30 days, preferably about 10 to 20 days.

[00136] As in step 1), a basal medium for use in culturing mammalian cells can be used as a culture medium. The medium is supplemented with ALK5 II, T3, LDN inhibitor, γ-secretase inhibitor XX, γ-secretase inhibitor RO, N-cysteine, an AXL inhibitor and ascorbic acid according to the previous report (Nature Biotechnology 2014; 32: 1121-1133) and can be suitably supplemented with a Wnt inhibitor, a ROCK inhibitor, FGF (preferably FGF2), a serum substitute, a vitamin, an antibiotic and the like. For example, the medium can be supplemented with ALK5 II, T3, LDN inhibitor, RO γ-secretase inhibitor and ascorbic acid or it can be supplemented with T3, ALK5 II inhibitor, ZnSO4, heparin, N-acetylcysteine, Trolox and R428 .

33/74

[00137] The culture can be performed by any two-dimensional culture and three-dimensional culture. The culture is performed by non-adherent culture without the use of feeder cells. For culture, a plate, flask, microplate, porous plate (Nunc), or similar, or a bioreactor is used. The culture vessel is preferably surface treated to decrease cell adhesion.

[00138] The temperature of the culture is not particularly limited and the culture is carried out at 30 to 40 ° C (for example, 37 ° C). The concentration of carbon dioxide in a culture vessel is on the order of, for example, 5%. Differentiation in pancreatic β cells

[00139] The cells obtained in the previous step can be induced to differentiate into pancreatic β cells. The stage of differentiation in a population of pancreatic β cells can be performed by transplanting a population of endocrine progenitor cells or a population of cells at a later stage of differentiation, preferably a population of insulin-producing cells, in a living body. of an animal.

[00140] "Animal" is preferably a mammal. Examples of these include humans, non-human primates, pigs, cattle, horses, sheep, goats, llamas, dogs, cats, rabbits, mice and guinea pigs. A human is preferred.

[00141] The transplant is preferably performed for an in vivo region where the cell population can be fixed in a certain position, and can be performed, for example, subcutaneously, intraperitoneally, for the peritoneal mesothelium, for the greater omentum, for a adipose tissue, for muscle tissue, or below the capsule of each organ, such as the pancreas or kidney, in the animal. The number of cells to be transplanted can vary depending on factors such as the stage of

34/74 differentiation of the cells to be transplanted, the age and body weight of a recipient, the size of a transplant site and the severity of a disease and is not particularly limited. For example, the number of cells can range from 10 × 104 cells to 10 × 1011 cells. The population of transplanted cells is induced to differentiate in an in vivo environment and can thus differentiate into the population of cells of interest, preferably a population of pancreatic β cells, which can then be recovered or can be inhabited in vivo as such .

[00142] For transplantation, the cell population can be incorporated into a gel containing alginic acid and then transplanted. For example, the population of cells embedded in the gel containing alginic acid can be enclosed in a device, such as a capsule, bag or chamber, and transplanted into a living body.

[00143] "Gel containing alginic acid" can be prepared according to a known approach (WO2010 / 032242 and WO2011 / 154941) and can be obtained by adding a crosslinking agent to a solution of alginate or alginic acid ester for gelation .

[00144] Alginate can be a water-soluble salt and a metal salt, an ammonium salt or the like can be used. For example, sodium alginate, calcium alginate or ammonium alginate can be used properly.

[00145] The alginic acid ester (also called propylene glycol alginate) is a derivative in which propylene glycol is attached to the carboxyl group of alginic acid through an ester bond.

[00146] The ratio of manuronic acid to guluronic acid (M / G ratio) contained in alginate is arbitrary. In general, in the case of M> G, a highly flexible gel can be formed. In the case of M <G, a strong gel can be formed. In the present invention, alginate containing guluronic acid can be used in a proportion of 10 to 90%, 20 to 80%, 30 to 70% or 40 to 60%.

35/74

[00147] The alginate or alginic acid ester can be contained in an amount of 0.05 to 10% by weight, preferably 0.1 to 5% by weight, more preferably 0.5 to 3% by weight, in one solvent. The solvent can be any solvent capable of dissolving alginate or alginic acid ester, and water, saline or the like can be used.

The crosslinking agent can be any crosslinking agent that can allow a solution of alginate or alginic acid ester to gel and is not particularly limited. A polyvalent metal cation can be used. The polyvalent metal cation is preferably a divalent metal cation, more preferably a calcium ion, a strontium ion or a barium ion. The cross-linking agent can be used in the form of a salt. In the present invention, at least one member selected from calcium chloride, strontium chloride and barium chloride can be used as the crosslinking agent.

[00149] The gel containing alginic acid may contain a nanofiber. Nanofiber is a natural or synthetic fiber with a diameter on the order of nanometers. Examples of natural nanofibers include nanofibers containing one or more of collagen, cellulose, silk fibroin, keratin, gelatin and polysaccharides, such as chitosan. Examples of synthetic nanofibers include polylactic acid (PLA), polycaprolactone (PCL), polyurethane (PU), poly (lactide-co-glycolide) (PLGA), poly (3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) and poly (ethylene) -co-vinylacetate) (PEVA). The nanofiber can be contained in an amount less than 1% by weight, for example, 0.9% by weight, 0.8% by weight, 0.7% by weight, 0.6% by weight, 0.5% by weight or less than the amount, in the gel containing alginic acid. The lower limit of the amount of nanofiber contained in the gel containing alginic acid is not particularly limited and can be 0.05% by weight or more, preferably 0.1% by weight or more.

[00150] In the present invention, "incorporation" means that a

36/74 population of endocrine progenitor cells or a population of cells at a later stage of differentiation is contained in a dispersed manner in the gel containing alginic acid.

[00151] The incorporation of the cell population in the gel containing alginic acid can be carried out by mixing the cell population in a solution of alginate or alginic acid ester and gelling it.

[00152] The population of cells can be contained in an amount selected from 1 × 104 cells to 1 × 109 cells / mL, preferably 1 × 107 cells to 1 × 108 cells / mL, in the alginate or alginic acid ester solution.

[00153] The gelation of the alginate solution or alginic acid ester containing the population of cells can be carried out by adding a cross-linking agent to the solution. The amount of the crosslinking agent added can be an amount selected from 0.1 to 5% by weight, for example, 0.1 to 1% by weight, based on the solution. Gelling can be carried out in a container with a predetermined configuration and / or shape for use in cell culture or cell transplantation, or in a mold designed to obtain a gel adapted to the container.

[00154] Alternatively, gelation can be carried out by forming a gel capsule containing alginic acid according to a known approach (WO2010 / 010902). Specifically, the alginate or alginic acid ester solution containing the cell population can be added dropwise to a solution of a crosslinking agent for gelation. The size of the liquid droplets can be adjusted according to the shape of a nozzle for dropwise addition or a dropwise addition method and, by extension, the size of the gel capsule containing alginic acid can be defined. The drip addition method is not particularly limited and can be carried out by an approach such as an air spray method, airless spray method or a static spray method.

37/74 The size of the gel capsule containing alginic acid is not particularly limited and may have a diameter of 5 mm or less, 1 mm or less, or 500 µm or less.

[00155] The crosslinking agent solution can contain the crosslinking agent in an amount selected from 0.1 to 10% by weight, for example, 0.1 to 5% by weight.

[00156] In the present invention, "FGFR1 inhibitor" is a substance with inhibitory activity at least for the fibroblast growth factor receptor (FGFR) 1. FGFR1 is a member of the four-pass transmembrane tyrosine kinase family (FGFR1 , FGFR2, FGFR3 and FGFR4), as a receptor with high affinity for growth factors FGF1 to FGF17. FGFR1 is involved in many signaling pathways associated with mesoderm pattern induction and pattern formation, cell proliferation and migration, organogenesis, bone growth, etc. The FGFR1 inhibitor used in the present invention is not particularly limited, as long as the FGFR1 inhibitor has FGFR1 inhibitory activity. The FGFR1 inhibitor can be a substance with FGFR1 inhibitory activity, as well as inhibitory activity of other FGFRs. In the present invention, "FGFR1 inhibitor" includes a substance with FGFR1 inhibitory activity, even if only slightly, and preferably refers to a substance that inhibits FGFR1 by 50% or more, more preferably a substance with an inhibitory concentration of 50% (IC50) of 1 µM or less, still preferably 100 nM or less, against FGFR1. A method for determining FGFR1 inhibitory activity can be selected from known methods. Examples include methods of determination using the EnzyChrom kinase test kit (BioAssay Systems). In the present invention, a conventionally known "FGFR1 inhibitor" can be used and can be verified in patent or non-patent literature. In the present invention, "FGFR1 inhibitor" can have at least

38/74 inhibitory activity of FGFR1 and may have inhibitory activity of other FGFRs. The "FGFR1 inhibitor" of the present invention can be, for example, an FGFR2 inhibitor, a FGFR3 inhibitor or a FGFR4 inhibitor, as long as it has FGFR1 inhibitory activity.

[00157] In the present invention, examples of the "FGFR1 inhibitor" include compounds given below (group of compounds D), as well as compounds with inhibitory activity for FGFR1 (or salts thereof) between compounds with structural formulas represented by the general formulas given below. The FGFR1 inhibitor is preferably a compound with a 50% inhibitory concentration (IC50) of 1 µM or less, more preferably 100 nM or less, against FGFR1 (or a salt thereof) between compound I and compound II with formulas structural elements represented by the following general formulas. (Compound I)

[00158] Examples of compound I include compounds represented by the following formula 1: [Formula 1] wherein the AB ring shows a bicyclic heterocycle with one or more substituent (s) (the AB ring can be a tricyclic heterocycle with one or more substituent (s)) or salts thereof.

[00159] Preferably, in the formula, the AB ring shows a nitrogen-containing bicyclic heterocycle with three substituents.

[00160] Examples of the substituents include substituents given below and substituents of the group of compounds D. The substituents of the group of

39/74 compounds D are preferred. (Compound II)

[00161] Examples of compound II include compounds represented by the following formula 2: [Formula 2] wherein ring D shows a 5- or 6-membered nitrogen-containing monocyclic heterocycle having optionally substituent (s); and one or more heterocycle (s) containing nitrogen other than ring D is contained or salts thereof.

[00162] Preferably, in the formula, the D ring shows an aromatic ring containing one or two nitrogen atoms and optionally having substituent (s).

[00163] Preferably, examples of one or more nitrogen-containing heterocycle (s), other than the D-ring, include piperazine optionally having substituent (s).

Examples of the substituents include substituents given below and substituents on the group of compounds D. Substituents on the group of compounds D are preferred.

[00165] As used herein, examples of the "heterocycle" include aromatic heterocycles and non-aromatic heterocycles each containing, as an atom constituting the ring in addition to the carbon atom, 1 to 4 hetero atoms selected from one nitrogen atom, one sulfur atom and an oxygen atom.

[00166] As used here, examples of the “aromatic heterocycle”

40/74 include an aromatic heterocycle of 5 to 14 members (preferably 5 to 10 members) containing, as an atom constituting the ring in addition to the carbon atom, 1 to 4 hetero atoms selected from a nitrogen atom, a sulfur atom and a oxygen atom. Preferable examples of the "aromatic heterocycle" include 5- or 6-membered monocyclic aromatic heterocycles such as thiophene, furan, pyrrole, imidazole, pyrazole, thiazole, isothiazole, oxazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, 1,2,4- oxadiazole, 1,3,4-oxadiazole, 1,2,4-thiadiazole, 1,3,4-thiadiazole, triazole, tetrazole, and triazine; and 8 to 14-membered fused polycyclic aromatic heterocycles (preferably bicyclic or tricyclic) such as benzothiophene, benzofuran, benzimidazole, benzoxazole, benzisoxazole, benzothiazole, benzisothiazole, benzotriazole, imidazopyridine, thienopyridine, pyridine, pyridazole imidazopyrimidine, thienopyrimidine, furopyrimidine, pyrrolpyrimidine, pyrazolpyrimidine, oxazolpyrimidine, thiazolpyrimidine, pyrazolpyrimidine, pyrazoltriazine, naphtho [2,3-b] thiophene, phenoxathine, indol, isoazole, isolinazole quinazoline, cinoline, carbazole, β-carboline, phenanthridine, acridine, phenazine, phenothiazine, and phenoxazine.

[00167] As used herein, examples of the "non-aromatic heterocycle" include a 3 to 14 membered (preferably 4 to 10 membered) non-aromatic heterocycle containing, as a ring constituent atom in addition to the carbon atom, 1 to 4 selected heteroatoms among a nitrogen atom, a sulfur atom and an oxygen atom. Preferable examples of the "non-aromatic heterocycle" include 3- to 8-membered monocyclic non-aromatic heterocycles such as aziridine, oxirane, thyrane, azetidine, oxetane, tiethane, tetrahydrothiophene, tetrahydrofuran, pyrroline, pyrrolidine, imidazoline, imidazolidine, oxazoline oxazolidine, pyrazoline, pyrazolidine, thiazoline, thiazolidine, tetrahydroisothiazole, tetrahydro-oxazole,

41/74 tetrahydroisoxazole, piperidine, piperazine, tetrahydropyridine, dihydropyridine, dihydrothiopyrane, tetrahydropyrimidine, tetrahydropyridazine, dihydropyran, tetrahydropyrane, tetrahydropyrine, diaphroline, morpholine, ti, morpholine, ti , azepine, azocane, diazocane, oxepan; and non-aromatic polycyclic heterocycles (preferably bi or tricyclic) fused from 9 to 14 members such as dihydrobenzofuran, dihydrobenzimidazole, dihydrobenzoxazole, dihydrobenzothiazole, dihydrobenzisothiazole, dihydronaphth [2,3-b] thiophene , tetrahydroisoquinoline, tetrahydroquinoline, 4H-quinolizine, indoline, isoindoline, tetrahydrothieno [2,3-c] pyridine, tetrahydrobenzazepine, tetrahydroxinoxaline, tetrahydrophenanthridine, hexahydrophenothiazine, hexahydrophen - hydrophthalazine, tetrahydronaphthyridine, tetrahydroquinazoline, tetrahydrocinoline, tetrahydrocarbazole, tetrahydro-β-carboline, tetrahydroacridine, tetrahydrophenazine, tetrahydrotioxanthene, octahydroisoquinoline.

[00168] As used herein, examples of the "nitrogen-containing heterocycle" include "heterocycles" containing at least one or more nitrogen atom (s) as a constituent atom of the ring.

[00169] In the following, each substituent used here will be defined in detail. Each substituent is as defined below unless otherwise specified.

[00170] As used herein, examples of the "halogen atom" include fluorine, chloride, bromine and iodine.

[00171] As used herein, examples of the "C1-6 alkyl group" include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, iso -hexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl and 2-ethylbutyl.

[00172] As used herein, examples of the "optionally halogenated C1-6 alkyl group" include a C1-6 alkyl group with optionally 1 to 7, preferably 1 to 5 halogen atoms. EXAMPLES

42/74 specifics include methyl, chloromethyl, difluoromethyl, trichloromethyl, trifluoromethyl, ethyl, 2-bromoethyl, 2,2,2-trifluoroethyl, tetrafluoroethyl, pentafluoroethyl, propyl, 2,2-difluoropropyl, 3,3,3-trifluoropropyl , isopropyl, butyl, 4,4,4-trifluorobutyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 5,5,5-trifluoropentyl, hexyl and 6,6,6-trifluorohexyl.

[00173] As used herein, examples of the "C2-6 alkenyl group" include ethylene, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 3-methyl -2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 4-methyl-3-pentenyl, 1-hexenyl, 3-hexenyl and 5-hexenyl.

[00174] As used herein, examples of the "C2-6 alkynyl group" include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl , 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl and 4-methyl-2-pentynyl.

[00175] As used herein, examples of the “C3-10 cycloalkyl group” include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicycle [2.2.1] heptila, bicycle [2.2.2] octyl , bicycles [3.2.1] octyl and adamantyl.

[00176] As used herein, examples of the "optionally halogenated C3-10 cycloalkyl group" include a C3-10 cycloalkyl group with optionally 1 to 7, preferably 1 to 5 halogen atoms. Specific examples thereof include cyclopropyl, 2,2-difluorocyclopropyl, 2,3-difluorocyclopropyl, cyclobutyl, difluorocyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

[00177] As used herein, examples of the "C3-10 cycloalkenyl group" include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl and cyclooctenyl.

[00178] As used herein, examples of the "C6-14 aryl group" include phenyl, 1-naphthyl, 2-naphthyl, 1-anthryl, 2-anthryl and 9-anthryl.

43/74

[00179] As used herein, examples of the "C7-16 aralkyl group" include benzyl, phenethyl, naphthylmethyl and phenylpropyl.

[00180] As used herein, examples of the "C1-6 alkoxy group" include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy and hexyloxy.

[00181] As used herein, examples of the "optionally halogenated C1-6 alkoxy group" include a C1-6 alkoxy group with optionally 1 to 7, preferably 1 to 5 halogen atoms. Specific examples thereof include methoxy, difluoromethoxy, trifluoromethoxy, ethoxy, 2,2,2-trifluoroethoxy, propoxy, isopropoxy, butoxy, 4,4,4-trifluorobutoxy, isobutoxy, sec-butoxy, pentyloxy and hexyloxy.

[00182] As used herein, examples of the "C3-10 cycloalkyloxy group" include cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, cycloheptyloxy and cyclooctyloxy.

[00183] As used herein, examples of the "C1-6 alkylthio group" include methylthio, ethylthio, propylthio, isopropylthio, butylthio, sec-butylthio, tert-butylthio, pentylthio and hexylthio.

[00184] As used herein, examples of the "optionally halogenated C1-6 alkylthio group" include a C1-6 alkylthio group with optionally 1 to 7, preferably 1 to 5 halogen atoms. Specific examples thereof include methylthio, difluoromethylthio, trifluoromethylthio, ethylthio, propylthio, isopropylthio, butylthio, 4,4,4-trifluorobutylthio, pentylthio and hexylthio.

[00185] As used herein, examples of the "C1-6 alkylcarbonyl group" include acetyl, propanoyl, butanoyl, 2-methylpropanoyl, pentanoyl, 3-methylbutanoyl, 2-methylbutanoyl, 2,2-dimethylpropanoyl, hexanoyl and heptanoyl.

[00186] As used herein, examples of the "optionally halogenated C1-6 alkylcarbonyl group" include a C1-6 alkylcarbonyl group with

44/74 optionally 1 to 7, preferably 1 to 5 halogen atoms. Specific examples thereof include acetyl, chloroacetyl, trifluoroacetyl, trichloroacetyl, propanoyl, butanoyl, pentanoyl and hexanoyl.

[00187] As used herein, examples of the "C1-6 alkoxycarbonyl group" include methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, sec-butoxycarbonyl, tert-butoxycarbonyl, pentyloxycarbonyl and hexyloxycarbonyl.

[00188] As used herein, examples of the "C6-14 aryl-carbonyl group" include benzoyl, 1-naphthyl and 2-naphthyl.

[00189] As used herein, examples of the "C7-16 aralkylcarbonyl group" include phenylacetyl and phenylpropionyl.

[00190] As used herein, examples of the "5- to 14-membered aromatic heterocyclylcarbonyl group" include nicotinoyl, isonicotinoyl, tenoyl and furoyl.

[00191] As used herein, examples of the "3- to 14-membered non-aromatic heterocyclylcarbonyl group" include morpholinylcarbonyl, piperidinylcarbonyl and pyrrolidinylcarbonyl.

[00192] As used herein, examples of the "mono- or di-C1-6 alkyl-carbamoyl group" include methylcarbamoyl, ethylcarbamoyl, dimethylcarbamoyl, diethylcarbamoyl and N-ethyl-N-methylcarbamoyl.

[00193] As used herein, examples of the "mono- or di-C7-16 aralkylcarbonyl group" include benzylcarbamoyl and phenethylcarbamoyl.

[00194] As used herein, examples of the "C1-6 alkylsulfonyl group" include methylsulfonyl, ethylsulfonyl, propylsulfonyl, isopropylsulfonyl, butylsulfonyl, sec-butylsulfonyl and tert-butylsulfonyl.

[00195] As used herein, examples of the "optionally halogenated C1-6 alkylsulfonyl group" include a C1-6 alkylsulfonyl group with optionally 1 to 7, preferably 1 to 5 halogen atoms. Specific examples of the same include methylsulfonyl, difluoromethylsulfonyl,

45/74 trifluoromethylsulfonyl, ethylsulfonyl, propylsulfonyl, isopropylsulfonyl, butylsulfonyl, 4,4,4-trifluorobutylsulfonyl, pentylsulfonyl and hexylsulfonyl.

[00196] As used herein, examples of the "C6-14 arylsulfonyl group" include phenylsulfonyl, 1-naphthylsulfonyl and 2-naphthylsulfonyl.

[00197] As used herein, examples of the "substituent" include a halogen atom, a cyano group, a nitro group, an optionally substituted hydrocarbon group, an optionally substituted heterocyclic group, an acyl group, an optionally substituted amino group, a group optionally substituted carbamoyl, an optionally substituted thiocarbamoyl group, an optionally substituted sulfamoyl group, an optionally substituted hydroxy group, an optionally substituted sulfanyl (SH) group and an optionally substituted silyl group.

[00198] In this specification, examples of the "hydrocarbon group" (including "optionally substituted" hydrocarbon group ") include a C1-6 alkyl group, a C2-6 alkenyl group, a C2-6 alkynyl group, a C3-10 cycloalkyl group, a C3-10 cycloalkenyl group, a C6-14 aryl group and a C7-16 aralkyl group.

[00199] In this specification, examples of the “optionally substituted hydrocarbon group” include a hydrocarbon group optionally with substituent (s) selected from the following substituent group A. [substituent group A]

[00200] (1) a halogen atom, (2) a nitro group, (3) a cyano group, (4) an oxo group, (5) a hydroxy group, (6) an optionally halogenated C1-6 alkoxy group , (7) a C6-14 aryloxy group (e.g., phenoxy, naphthoxy),

46/74

(8) a C7-16 aralkyloxy group (for example, benzyloxy), (9) an aromatic heterocyclyl group of 5 to 14 members (for example, pyridyloxy), (10) a non-aromatic heterocyclyl group of 3 to 14 members (for example, morpholinyloxy, piperidinyloxy), (11) a C1-6 alkylcarbonyloxy group (for example, acetoxy, propanoyloxy), (12) a C6-14 arylcarbonyloxy group (for example, benzoyloxy, 1-naphthyloxy, 2- naphthoyloxy), (13) a C1-6 alkoxycarbonyloxy group (for example, methoxycarbonyloxy, ethoxycarbonyloxy, propoxycarbonyloxy, butoxycarbonyloxy), (14) a mono- or di-C1-6 alkylcarbamoyloxy group (for example, methylcarbamoyloxy, ethyl) , dimethylcarbamoyloxy, diethylcarbamoyloxy), (15) a C6-14 aryl-carbamoyloxy group (eg, phenylcarbamoyloxy, naphthylcarbamoyloxy), (16) an aromatic heterocyclylcarbonyloxy group of 5 to 14 members (eg, nicotinoyloxy), (17) non-aromatic 3- to 14-membered heterocyclylcarbonyloxy (eg, morpholinylcarbonyloxy, piperidinylcarb onyloxy), (18) an optionally halogenated C1-6 alkylsulfonyloxy group (eg, methylsulfonyloxy, trifluoromethylsulfonyloxy), (19) a C6-14 arylsulfonyloxy group optionally substituted with a C1-6 alkyl group (eg, phenylsulfonyloxy, toluenesulfonyl) (20) an optionally halogenated C1-6 alkylthio group, (21) a 5- to 14-membered aromatic heterocyclic group, (22) a 3- to 14-membered non-aromatic heterocyclic group, (23) a formyl group, (24) a carboxy group,

47/74

(25) an optionally halogenated C1-6 alkylcarbonyl group, (26) an arylcarbonyl C6-14 group, (27) a 5- to 14-membered aromatic heterocyclylcarbonyl group, (28) a 3- to non-aromatic heterocyclylcarbonyl group 14 members, (29) a C1-6 alkoxycarbonyl group, (30) a C6-14 aryloxycarbonyl group (e.g., phenyloxycarbonyl, 1-naphthyloxycarbonyl, 2-naphthyloxycarbonyl), (31) a C7-16 aralkyloxy group -carbonyl (for example, benzyloxycarbonyl, phenethyloxycarbonyl), (32) a carbamoyl group, (33) a thiocarbamoyl group, (34) a mono- or di-C1-6 alkyl-carbamoyl group, (35) a C6-14 group aryl-carbamoyl (for example, phenylcarbamoyl), (36) an aromatic 5- to 14-membered heterocyclylcarbamoyl group (eg, pyridylcarbamoyl, thienylcarbamoyl), (37) a 3 to 14-membered non-aromatic heterocyclyl carbamoyl group (eg, morpholinylcarbamoyl piperidinylcarbamoyl), (38) an optionally halogenated C1-6 alkylsulfonyl group, (39) a C6-14 arylsulfonyl group, (4 0) a 5- to 14-membered aromatic heterocyclylsulfonyl group (e.g., pyridylsulfonyl, thienylsulfonyl), (41) an optionally halogenated C1-6 alkylsulfinyl group, (42) a C6-14 arylsulfinyl group (eg, phenylsulfinyl, 1-naphthylsulfinyl , 2-naphthylsulfinyl), (43) an aromatic heterocyclyl sulfinyl group of 5 to 14

48/74 members (for example, pyridylsulfinyl, thienylsulfinyl), (44) an amino group, (45) a mono- or di-C1-6 alkylamino group (for example, methylamino, ethylamino, propylamino, isopropylamino, butylamino, dimethylamino, diethylamino, dipropylamino, dibutylamino, N-ethyl-N-methylamino), (46) a mono- or di-C6-14 arylamino group (for example, phenylamino), (47) an aromatic heterocyclyl group of 5 to 14 members (for pyridylamino), (48) a C7-16 aralkylamino group (for example, benzylamino), (49) a formylamino group, (50) a C1-6 alkylcarbonylamino group (for example, acetylamino, propanoylamino, butanoylamino), (51) a (C1-6 alkyl) (C1-6 alkylcarbonylamino group (for example, N-acetyl-N-methylamino), (52) a C6-14 arylcarbonylamino group (for example, phenylcarbonylamino, naphthylcarbonylamino) , (53) a C1-6 alkoxycarbonylamino group (e.g., methoxycarbonylamino, ethoxycarbonylamino, propoxycarbonylamino, butoxycarbonylamino, tert-butoxycarbonylamino), (54) a C group 7-16 aralkyloxycarbonylamino (eg, benzyloxycarbonylamino), (55) a C1-6 alkylsulfonylamino group (eg, methylsulfonylamino, ethylsulfonylamino), (56) a C6-14 arylsulfonylamino group optionally substituted with a C1-6 alkyl group ( for example, phenylsulfonylamino, toluenesulfonylamino),

49/74 (57) an optionally halogenated C1-6 alkyl group, (58) a C2-6 alkenyl group, (59) a C2-6 alkynyl group, (60) a C3-10 cycloalkyl group, (61) a group C3-10 cycloalkenyl and (62) a C6-14 aryl group.

[00201] The number of the substituents mentioned above in the "optionally substituted hydrocarbon group" is, for example, 1 to 5, preferably 1 to 3. When the number of the substituents is two or more, the respective substituents can be the same or different.

[00202] In this specification, examples of the “heterocyclic group” (including “optionally substituted heterocyclic group”) include (i) an aromatic heterocyclic group, (ii) a non-aromatic heterocyclic group and (iii) a non-aromatic heterocyclic group and (iii) a non-aromatic heterocyclic group and (iii) 7 to 10-membered bridged heterocyclic group, each containing, as a ring constituent atom in addition to the carbon atom, 1 to 4 heteroatoms selected from a nitrogen atom, a sulfur atom and an oxygen atom.

[00203] In this specification, examples of the "aromatic heterocyclic group" (including "5- to 14-membered aromatic heterocyclic group") include a 5- to 14-membered aromatic heterocyclic group (preferably 5 to 10 members) containing, as an atom constituent of the ring in addition to the carbon atom, 1 to 4 heteroatoms selected from a nitrogen atom, a sulfur atom and an oxygen atom.

[00204] Preferable examples of the "aromatic heterocyclic group" include aromatic heterocyclic groups monocyclic 5- or 6-members such as thienyl, furyl, pyrrolyl, imidazolyl, pirazolila, thiazolyl, isotiazolila, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, 1 , 2,4- oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl,

50/74 triazolyl, tetrazolyl, triazinyl and the like; and polycyclic aromatic heterocyclic groups (preferably bi or tricyclic) cast from 8 to 14 members such as benzotiofenila, benzofuranyl, benzimidazolila, benzoxazolila, benzisoxazolila, benzotiazolila, benzisotiazolila, benzotriazolila, imidazopiridinila, tienopiridinila, furopiridinila, pirrolpiridinila, pirazolpiridinila, oxazolpiridinila, tiazolpiridinila, imidazopirazinila , imidazopyrimidinyl, thienopyrimidinyl, furopyrimidinyl, pyrrolpyrimidinyl, pyrazolpyrimidinyl, oxazolpyrimidinyl, thiazolpyrimidinyl, pyrazoltriazinyl, naphtho [2,3-b] thienyl, phenoxyinyl, quinol, isol, indolyl, isol, isol, , cinolinyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl and the like.

[00205] In this specification, examples of the "non-aromatic heterocyclic group" (including "3-14 membered non-aromatic heterocyclic group") include a 3-14 membered (preferably 4-10 membered) non-aromatic heterocyclic group containing, as an atom constituting the ring in addition to the carbon atom, 1 to 4 heteroatoms selected from a nitrogen atom, a sulfur atom and an oxygen atom.

[00206] Preferable examples of the "non-aromatic heterocyclic group" include monocyclic non-aromatic heterocyclic groups of 3 to 8 membered aziridinila such as, oxiranila, tiiranila, azetidinyl, oxetanila, tietanila, tetra- hidrotienila, hidrofuranila tetrahydropyranyl, pyrrolinyl, pyrrolidinyl, imidazolinila , imidazolidinyl, oxazolinyl, oxazolidinyl, pyrazolinyl, pyrazolidinyl, thiazolinyl, thiazolidinyl, tetrahydroisothiazolyl, tetrahydro-oxazolyl, tetrahydro-oxazolyl, piperidinyl, piperazinyl, tetrahydro-pyridine, hydropyridine, dihydro-pyridine, hydro-pyridine, hydropyridine , tetrahydropyridazinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, azepanyl, diazepanyl, azepinyl, oxepanyl,

51/74 azocanil, diazocanil and the like; and polycyclic non-aromatic heterocyclic groups (preferably 9 to 14-membered bicyclic or tricyclic groups such as dihydrobenzofuranyl, dihydrobenzimidazolyl, dihydrobenzoxazolyl, dihydrobenzothiazolyl, dihydrobenzisothiazolyl, dihydronapto [2,3-b] tien , tetrahydroisoquinolyl, tetrahydroquinolyl, 4H-quinolizinyl, indolinyl, isoindolinyl, tetrahydrothieno [2,3-c] pyridinyl, tetrahydrobenzazepinyl, tetrahydroxyninhydrin, tetrahydrophenanthridine, hexahydrin, hexahydrophenothen -hydrophthalazinyl, tetrahydronaphthyridinyl, tetrahydroquinazolinyl, tetrahydrocinolinyl, tetrahydrocarbazolyl, tetrahydro-β-carbolinyl, tetrahydroacridinyl, tetrahydrophenazinyl, tetrahydrotioxanthenyl, octahydroxyquinol and similar.

[00207] In this specification, preferable examples of the "7 to 10 membered heterocyclic bridging group" include quinuclidinyl and 7-azabicyclo [2.2.1] heptanyl.

[00208] In this specification, examples of the "nitrogen-containing heterocyclic group" include a "heterocyclic group" containing at least one nitrogen atom as a ring constituent atom.

[00209] In the present specification, examples of the "optionally substituted heterocyclic group" include a heterocyclic group with optionally substituent (s) selected from the aforementioned substituent group A.

[00210] The number of the substituents in the "optionally substituted heterocyclic group" is, for example, 1 to 3. When the number of the substituents is two or more, the respective substituents can be the same or different.

[00211] In this specification, examples of the "acyl group" include a formyl group, a carboxy group, a carbamoyl group, a thiocarbamoyl group, a sulfine group, a sulfo group, a sulfamoyl group

52/74 and a phosphono group, each optionally with “1 or 2 substituents selected from a C1-6 alkyl group, a C2-6 alkenyl group, a C3-10 cycloalkyl group, a C3-10 cycloalkenyl group, a C6-14 aryl group, C7-16 aralkyl group, 5 to 14 membered aromatic heterocyclic group and 3 to 14 membered non-aromatic heterocyclic group, each of which optionally has 1 to 3 substituents selected from a halogen atom , an optionally halogenated C1-6 alkoxy group, a hydroxy group, a nitro group, a cyano group, an amino group and a carbamoyl group ”.

[00212] Examples of the "acyl group" also include a hydrocarbon-sulfonyl group, a heterocyclylsulfonyl group, a hydrocarbon-sulfinyl group and a heterocyclylsulfinyl group.

[00213] Here, the hydrocarbon-sulfonyl group means a sulfonyl group attached to the hydrocarbon group, the heterocyclyl sulfonyl group means a sulfonyl group attached to the heterocyclic group, the hydrocarbon-sulfinyl group means a sulfinyl group attached to the hydrocarbon group and the heterocyclyl sulfinyl group sulfinyl group attached to the heterocyclic group.

[00214] Preferable examples of the "acyl group" include a formyl group, a carboxy group, a C1-6 alkylcarbonyl group, a C2-6 alkenylcarbonyl group (e.g., crotonoyl), a C3-10 cycloalkyl- carbonyl (for example, cyclobutanocarbonyl, cyclopentanocarbonyl, cyclohexanocarbonyl, cycloheptanocarbonyl), a C3-10 cycloalkenylcarbonyl group (eg 2-cyclohexenocarbonyl), a C6-14 arylcarbonyl group, a C7- 16 aralkylcarbonyl, a 5- to 14-membered aromatic heterocyclylcarbonyl group, a 3- to 14-membered non-aromatic heterocyclylcarbonyl group, a C1-6 alkoxycarbonyl group, a C6-14 aryloxycarbonyl group (for example, phenyloxycarbonyl, naphthyloxycarbonyl ), a C7-16 aralkyloxycarbonyl group (e.g. benzyloxycarbonyl, phenylethyloxycarbonyl),

53/74 a carbamoyl group, a mono- or di-C1-6 alkyl-carbamoyl group, a mono- or di-C2-6 alkenyl-carbamoyl group (for example, diallylcarbamoyl), a mono- or di-C3- 10 cycloalkyl-carbamoyl (for example, cyclopropylcarbamoyl), a mono- or di-C6-14 aryl-carbamoyl group (eg, phenylcarbamoyl), a mono- or di-C7-16 aralkyl-carbamoyl group, an aromatic heterocyclyl carbamoyl group 5 to 14 members (for example, pyridylcarbamoyl), a thiocarbamoyl group, a mono- or di-C1-6 alkyl-thiocarbamoyl group (for example, methylthiocarbamoyl, N-ethyl-N-methylthiocarbamoyl), a mono- or di- C2-6 alkenyl-thiocarbamoyl (for example, diallylthiocarbamoyl), a mono- or di-C3-10 cycloalkyl-thiocarbamoyl (for example, cyclopropylthiocarbamoyl, cyclohexylthiocarbamoyl), a mono- or di-C6-14 aryl-thiocarbamoyl group (for example, phenylthiocarbamoyl), a mono- or di-C7-16 aralkyl-thiocarbamoyl group (for example, benzylthiocarbamoyl, phenethylthiocarbamoyl), a group 5- to 14-membered aromatic heterocyclylthiocarbamoyl (eg, pyridylthiocarbamoyl), a sulfin group, a C1-6 alkylsulfinyl group (eg, methylsulfinyl, ethylsulfinyl), a sulpho group, a C1-6 alkylsulfonyl group, a C6-14 arylsulfonyl group , a phosphono group and a mono- or di-C1-6 alkylphosphono group (e.g., dimethylphosphono, diethylphosphono, diisopropylphosphono, dibutylphosphono).

[00215] In this specification, examples of the "optionally substituted amino group" include an amino group with optionally "1 or 2 substituents selected from a C1-6 alkyl group, a C2-6 alkenyl group, a C3-10 group cycloalkyl, a C6-14 aryl group, a C7-16 aralkyl group, a C1-6 alkylcarbonyl group, a C6-14 arylcarbonyl group, a C7-16 aralkylcarbonyl group, an aromatic heterocyclylcarbonyl group from 5 to 14 members, a 3- to 14-membered non-aromatic heterocyclylcarbonyl group, a C1-6 alkoxycarbonyl group, a 5- to 14-membered aromatic heterocyclic group, a carbamoyl group, a mono- or di-C1-6 alkyl-

54/74 carbamoyl, a mono- or di-C7-16 aralkyl-carbamoyl group, a C1-6 alkylsulfonyl group and a C6-14 arylsulfonyl group, each of which optionally has 1 to 3 substituents selected from the substituent group THE".

Preferred examples of the optionally substituted amino group include an optionally halogenated amino group, a mono- or di- (C1-6 alkyl) amino group (e.g., methylamino, trifluoromethylamino, dimethylamino, ethylamino, diethylamino, propylamino, dibutylamino), a mono- or di-C2-6 alkenylamino group (for example, diallylamino), a mono- or di-C3-10 cycloalkylamino group (for example, cyclopropylamino, cyclohexylamino), a mono- or di-C6-14 group arylamino (for example, phenylamino), a mono- or di-C7-16 aralkylamino group (for example, benzylamino, dibenzylamino), a mono- or di- (C1-6 alkyl) - optionally halogenated carbonylamino group (for example, acetylamino , propionylamino), a mono- or di-C6-14 aryl-carbonylamino group (for example, benzoylamino), a mono- or di-C7-16 aralkyl-carbonylamino group (for example, benzylcarbonylamino), a mono- or di-group - 5- to 14-membered aromatic heterocyclylcarbonylamino (eg nicotinoylamino, isonicotinoylamino), a group non-aromatic 3- to 14-membered mono- or di-heterocyclylcarbonylamino (eg, piperidinylcarbonylamino), a mono- or di-C1-6 alkoxycarbonylamino group (eg, tert-butoxycarbonylamino), an aromatic heterocyclyl group of 5 to 14 members (for example, pyridylamino), a carbamoylamino group, a group (mono- or di-C1-6 alkyl-carbamoyl) amino (for example, methylcarbamoylamino), a group (mono- or di-C7-16 aralkyl-carbamoyl) amino (for example, benzylcarbamoylamino), a C1-6 alkylsulfonylamino group (for example, methylsulfonylamino, ethylsulfonylamino), a C6-14 arylsulfonylamino group (for example, phenylsulfonylamino), a (C1-6 alkyl) group (C1-6 alkyl- carbonyl) amino (e.g., N-acetyl-N-methylamino)

55/74 and a (C1-6 alkyl) (C6-14 aryl-carbonyl) amino group (e.g., N-benzoyl-N-methylamino).

[00217] In this specification, examples of the "optionally substituted carbamoyl group" include a carbamoyl group with optionally "1 or 2 substituents selected from a C1-6 alkyl group, a C2-6 alkenyl group, a C3-10 group cycloalkyl, a C6-14 aryl group, a C7-16 aralkyl group, a C1-6 alkylcarbonyl group, a C6-14 arylcarbonyl group, a C7-16 aralkylcarbonyl group, an aromatic heterocyclylcarbonyl group from 5 to 14 members, a 3- to 14-membered non-aromatic heterocyclylcarbonyl group, a C1-6 alkoxycarbonyl group, a 5- to 14-membered aromatic heterocyclic group, a carbamoyl group, a mono- or di-C1-6 alkyl-carbamoyl group and a mono- or di-C7-16 aralkyl carbamoyl group, each of which optionally has 1 to 3 substituents selected from the A ”substituent group.

Preferred examples of the optionally substituted carbamoyl group include a carbamoyl group, a mono- or di-C1-6 alkyl-carbamoyl group, a mono- or di-C2-6 alkenyl-carbamoyl group (e.g., diallyl-carbamoyl), a mono- or di-C3-10 cycloalkyl-carbamoyl group (for example, cyclopropylcarbamoyl, cyclohexylcarbamoyl), a mono- or di-C6-14 aryl-carbamoyl group (for example, phenylcarbamoyl), a mono- or di- C7-16 aralkyl-carbamoyl, a mono- or di-C1-6 alkyl-carbonyl-carbamoyl group (for example, acetylcarbamoyl, propionylcarbamoyl), a mono- or di-C6-14 aryl-carbonyl-carbamoyl group (for example, benzoylcarbamoyl) and a 5- to 14-membered aromatic heterocyclylcarbamoyl group (eg, pyridylcarbamoyl).

[00219] In this specification, examples of the "optionally substituted thiocarbamoyl group" include a thiocarbamoyl group with optionally "1 or 2 substituents selected from a C1-6 alkyl group, a C2-6 alkenyl group, a C3-10 group cycloalkyl, a group

56/74 C6-14 aryl, a C7-16 aralkyl group, a C1-6 alkylcarbonyl group, a C6-14 arylcarbonyl group, a C7-16 aralkylcarbonyl group, an aromatic heterocyclylcarbonyl group from 5 to 14 members, a 3- to 14-membered non-aromatic heterocyclylcarbonyl group, a C1-6 alkoxycarbonyl group, a 5- to 14-membered aromatic heterocyclic group, a carbamoyl group, a mono- or di-C1-6 alkyl-carbamoyl group and a mono- or di-C7-16 aralkyl carbamoyl group, each of which optionally has 1 to 3 substituents selected from the A ”substituent group.

Preferred examples of the optionally substituted thiocarbamoyl group include a thiocarbamoyl group, a mono- or di-C 1-6 alkyl-thiocarbamoyl group (e.g., methylthiocarbamoyl, ethylthiocarbamoyl, dimethylthiocarbamoyl, diethyltiocarbamoyl, N-ethylamylamyl, N-ethylamyl-N-ethyl-N-ethylamino-N-ethyl-carbamoyl) mono- or di-C2-6 alkenyl-thiocarbamoyl group (eg, diallylthiocarbamoyl), a mono- or di-C3-10 cycloalkyl-thiocarbamoyl group (eg, cyclopropylthiocarbamoyl, cyclohexylthiocarbamoyl), a mono- or di- C6-14 aryl-thiocarbamoyl (for example, phenylthiocarbamoyl), a mono- or di-C7-16 aralkyl-thiocarbamoyl (for example, benzylthiocarbamoyl, phenethylthiocarbamoyl), a mono- or di-C1-6 alkyl-carbonyl-thiocarbamoyl group (e.g., acetylthiocarbamoyl, propionylthiocarbamoyl), a mono- or di-C6-14 aryl-carbonyl-thiocarbamoyl group (e.g., benzoylthiocarbamoyl) and an aromatic 5- to 14-membered heterocyclylthiocarbamoyl group (e.g., pyridylthiocarbamoyl).

[00221] In this specification, examples of the "optionally substituted sulfamoyl group" include a sulfamoyl group with optionally "1 or 2 substituents selected from a C1-6 alkyl group, a C2-6 alkenyl group, a C3-10 group cycloalkyl, a C6-14 aryl group, a C7-16 aralkyl group, a C1-6 alkyl carbonyl group, a C6-14 aryl carbonyl group, a C7-16 aralkyl carbonyl group, a group

57/74 aromatic heterocyclylcarbonyl of 5 to 14 members, a non-aromatic heterocyclylcarbonyl group of 3 to 14 members, a C1-6 alkoxycarbonyl group, an aromatic heterocyclic group of 5 to 14 members, a carbamoyl group, a mono- or di-C1-6 alkyl-carbamoyl and a mono- or di-C7-16 aralkyl-carbamoyl group, each of which optionally has 1 to 3 substituents selected from the A ”substituent group.

Preferred examples of the optionally substituted sulfamoyl group include a sulfamoyl group, a mono- or di-C1-6 alkylsulfamoyl group (e.g., methylsulfamoyl, ethylsulfamoyl, dimethylsulfamoyl, diethylsulfamoyl, N-ethyl-N-methylsulfamoyl), one mono- or di-C2-6 alkenyl sulfamoyl group (for example, diallylsulfamoyl), a mono- or di-C3-10 cycloalkyl-sulfamoyl group (for example, cyclopropylsulfamoyl, cyclohexylsulfamoyl), a mono- or di- C6-14 aryl-sulfamoyl (eg, phenylsulfamoyl), a mono- or di-C7-16 aralkyl-sulfamoyl group (eg, benzylsulfamoyl, phenethylsulfamoyl), a mono- or di-C1-6 alkyl-carbonyl-sulfamoyl group (eg, acetylsulfamoyl, propionylsulfamoyl), a mono- or di-C6-14 aryl-carbonyl-sulfamoyl group (eg, benzoylsulfamoyl) and an aromatic 5- to 14-membered heterocyclyl sulfamoyl group (eg, pyridylsulfamoyl).

[00223] In this specification, examples of the “optionally substituted hydroxy group” include a hydroxyl group with optionally “a substituent selected from a C1-6 alkyl group, a C2-6 alkenyl group, a C3-10 cycloalkyl group, a C6-14 aryl group, a C7-16 aralkyl group, a C1-6 alkylcarbonyl group, a C6-14 arylcarbonyl group, a C7-16 aralkylcarbonyl group, an aromatic heterocyclylcarbonyl group of 5 to 14 members , a 3- to 14-membered non-aromatic heterocyclylcarbonyl group, a 5- to 14-membered C1-6 alkoxycarbonyl group, a 5- to 14-membered aromatic heterocyclic group, a carbamoyl group, a mono- or di-C1-6 alkyl carbamoyl group, a mono- or di-C7-16 aralkyl carbamoyl group, a C1-6 group

58/74 alkylsulfonyl and a C6-14 arylsulfonyl group, each of which optionally has 1 to 3 substituents selected from the A ”substituent group.

Preferred examples of the optionally substituted hydroxy group include a hydroxy group, a C1-6 alkoxy group, a C2-6 alkenyloxy group (e.g., allyloxy, 2-butenyloxy, 2-pentenyloxy, 3-hexenyloxy), a C3 group -10 cycloalkyloxy (eg, cyclohexyloxy), a C6-14 aryloxy group (eg, phenoxy, naphthyloxy), a C7-16 aralkyloxy group (eg, benzyloxy, phenethyloxy), a C1-6 alkylcarbonyloxy group (for example, acetyloxy, propionyloxy, butyryloxy, isobutyryloxy, pivaloyloxy), a C6-14 aryl-carbonyloxy group (for example, benzoyloxy), a C7-16 aralkyl-carbonyloxy group (for example, benzylcarbonyloxy), a heterocyclic carbonyloxycarbonyloxy group 5 to 14 members (for example, nicotinoyloxy), a 3- to 14-membered non-aromatic heterocyclylcarbonyloxy group (for example, piperidinylcarbonyloxy), a C1-6 alkoxycarbonyloxy group (for example, tert-butoxycarbonyloxy), an aromatic heterocyclyloxy group 5 to 14 members (for example, pyridyloxy), a Carba group moyloxy, a C1-6 alkyl-carbamoyloxy group (eg, methylcarbamoyloxy), a C7-16 aralkyl-carbamoyloxy group (eg, benzylcarbamoyloxy), a C1-6 alkylsulfonyloxy group (eg, methylsulfonyloxy, ethylsulfonyloxy) and a group -14 arylsulfonyloxy (for example, phenylsulfonyloxy).

[00225] In this specification, examples of the "optionally substituted sulfanyl group" include a sulfanyl group with optionally "a substituent selected from a C1-6 alkyl group, a C2-6 alkenyl group, a C3-10 cycloalkyl group, a C6-14 aryl group, a C7-16 aralkyl group, a C1-6 alkylcarbonyl group, a C6-14 arylcarbonyl group and an aromatic heterocyclic group of 5 to 14 members, each of which optionally has 1 to 3 substituents selected from the group

59/74 substituent A ”and a halogenated sulfanyl group.

Preferred examples of the optionally substituted sulfanyl group include a sulfanyl group (-SH), a C1-6 alkylthio group, a C2-6 alkenyl thio group (for example, allyl, 2-butenylthio, 2-pentenylthio, 3-hexenylthio) , a C3-10 cycloalkylthio group (for example, cyclohexylthio), a C6-14 arylthio group (for example, phenylthio, naphthylthio), a C7-16 aralkylthio group (for example, benzylthio, phenethylthio), a C1- group 6 alkyl-carbonylthio (for example, acetylthio, propionylthio, butyrylthio, isobutyrylthio, pivaloylthio), a C6-14 aryl-carbonylthio group (for example, benzoylthio), an aromatic heterocyclyl group of 5 to 14 members (for example, pyridylthio) and a halogenated thio group (e.g., pentafluorothio).

[00227] In this specification, examples of the “optionally substituted silyl group” include a silyl group with optionally “1 to 3 substituents selected from a C1-6 alkyl group, a C2-6 alkenyl group, a C3-10 group cycloalkyl, a C6-14 aryl group and a C7-16 aralkyl group, each of which optionally has 1 to 3 substituents selected from substituent group A ”.

Preferable examples of the optionally substituted silyl group include a tri-C 1-6 alkylsilyl group (e.g., trimethylsilyl, tert-butyl (dimethyl) silyl).

[00229] More specifically, examples of the FGFR1 inhibitor that can be used in the present invention include PD-166866 (1- [2-amino-6- (3,5-dimethoxyphenyl) -pyrido (2,3-d) pyrimidin -7-il] -3-tert-butylurea: CAS nº: 192705-79-6) (in this specification, this compound is also called “CAS192705-79-6”), E-3810 (CAS nº: 1058137 -23-7), PD- 173074 (CAS No.: 219580-11-7), FGFR4-IN-1 (CAS No.: 1708971-72-5), FGFR-IN-1 (CAS No.: 1448169-71-8 ), FIIN-2 (CAS No.: 1633044-56-0), AZD4547 (CAS No.: 1035270-39-3), FIIN-3 (CAS No.: 1637735-84-2), NVP- BGJ398 (CAS No.: 1310746 -10-1), NVP-BGJ398 (CAS no .: 872511-34-7),

60/74

CH5183284 (CAS No.: 1265229-25-1), Derazantinib (CAS No.: 1234356-69- 4), Derazantinib Racemate, Ferulic Acid (CAS No.: 1135-24-6), SSR128129E (CAS No.: 848318-25- 2), free acid SSR128129E (CAS No.: 848463-13-8), Erdafitinib (CAS No.: 1346242-81-6), BLU9931 (CAS No.: 1538604-68-0), PRN1371 (CAS No.: 1802929-43- 6), S49076 (CAS No.: 1265965-22-7), LY2874455 (CAS No.: 1254473-64-7), Linsitinib (CAS No.: 867160-71-2), Dovitinib (CAS No.: 405169-16-6) , Anlotinib (CAS number: 1058156-90-3), Brivanibe (CAS number: 649735-46-6), Derazantinib (CAS number: 1234356-69-4), Anlotinib dihydrochloride (CAS number: 1360460-82-7) , ACTB- 1003 (CAS No.: 939805-30-8), BLU-554 (CAS No.: 1707289-21-1), Rogaratinibe (CAS No.: 1443530-05-9), BIBF 1120 esilate (CAS No.: 656247- 18-6), hydrochloride TG 100572 (CAS nº: 867331-64-4), ENMD-2076 (CAS nº: 934353-76-1), Alanine de Brivanibe (CAS nº: 649735-63-7), TG 100572 ( CAS No.: 867334-05-2), BIBF 1120 (CAS No.: 656247-17-5), ENMD-2076 tartrate (CAS No.: 1291074-87-7), TSU-68 (CA No. S: 252916-29-3), Ponatinib (CAS No.: 943319-70-8), Sulfatinib (CAS No.: 1308672-74-3), LY2784544 (CAS No.: 1229236-86-5), Dovitinib Lactate (No. CAS: 692737-80-7), SU 5402 (CAS No.: 215543-92-3), FGF-401 (CAS No.: 1708971-55-4), tyrosine kinase- IN-1 (CAS No.: 705946-27- 6), PP58 (CAS No.: 212391-58-7), TG 100801 hydrochloride (CAS No.: 1018069-81-2), Crenolanib (CAS No.: 670220-88-9), TG 100801 (CAS No.: 867331-82 -6), Pazopanibe Hydrochloride (CAS No.: 635702- 64-6), Pazopanibe (CAS No.: 444731-52-6), PD168393 (CAS No.: 194423-15-9), Apatinibe (CAS No.: 1218779-75 -9), Palbocyclibibe isethionate (CAS number: 827022-33-3), Foretinibe (CAS number: 849217-64-7), Lenvatinibe (CAS number: 417716-92-8), Tandutinib (CAS number: 387867-13-13 -2), and their salts (these compounds are called the group of compounds D). Each of these compounds can have one or more substituent (s) selected from those described above, provided that the compound has FGFR1 inhibitory activity, preferably a 50% inhibitory concentration (IC50) of 100 nM or

61/74 lower against FGFR1.

[00230] The substructure (substituent, ring, etc.) of each of these compounds can be partially converted, provided that the compound has FGFR1 inhibitory activity, preferably a 50% inhibitory concentration (IC50) of 100 nM or less against FGFR1 .

[00231] In the present invention, the FGFR1 inhibitor is preferably CAS192705-79-6 (1- [2-amino-6- (3,5-dimethoxyphenyl) -pyrido (2,3-d) pyrimidin-7-yl] -3-tert-butylurea: CAS No.: 192705-79-6), E-3810 (CAS No.: 1058137-23-7), or PD173074 (CAS No.: 219580-11-7).

[00232] CAS192705-79-6 was found to have inhibitory activity for FGFR1, FGFR2, FGFR3 and FGFR4 by the following test: fluorescent probes binding to FGFR1, FGFR2, FGFR3 and FGFR4 prepared as recombinant proteins were authorized to act in the presence or absence of CAS192705-79-6, and fluorescent signals were detected using a plate reader. The inhibitory activity of CAS192705-79-6 for each protein (pIC50) was calculated from the change in the fluorescent signal between the presence and absence of CAS192705-79-6.

[00233] The FGFR1 inhibitor is not limited to the compounds described above, and an antisense oligonucleotide or siRNA against FGFR1 mRNA, a FGFR1 binding antibody, a dominant negative FGFR1 mutant or the like can also be used as the FGFR1 inhibitor . This FGFR1 inhibitor is either commercially available or can be synthesized according to a known method.

[00234] Each compound mentioned above or a salt thereof can be used as the FGFR1 inhibitor. The amount of EGFR1 inhibitor added to the medium is determined appropriately according to the compound or the salt thereof used and is generally about 0.00001 µM to 100 µM, preferably 0.01 µM to 10 µM.

[00235] The population of endocrine progenitor cells or the

62/74 cell population at a later stage of differentiation can be treated with the FGFR1 inhibitor by the coexistence of the cell population with the FGFR1 inhibitor. For example, treatment can be performed by culturing the cell population in a medium supplemented with the FGFR1 inhibitor. The FGFR1 inhibitor can be contained in any amount capable of inhibiting FGFR1 activity in the medium and can be contained in an amount of, for example, 10 µM or less or 5 µM or less, preferably in an amount less than 5 µM, less than 4 µM, less than 3 µM or less than 2 µM. The lower limit of the amount of FGFR1 inhibitor added is not particularly limited and can be 0.1 µM or more, preferably 0.5 µM or more. The amount of FGFR1 inhibitor added is preferably less than 5 µM and 0.1 µM or more, more preferably less than 5 µM and 0.5 µM or more. Culture in the presence of the FGFR1 inhibitor can be performed for at least 12 hours, preferably 24 hours or more, 2 days or more, 4 days or more, 8 days or more, 10 days or more, or 15 days or more. Culture in the presence of the FGFR1 inhibitor is preferably carried out for 4 days or more. The medium can be replaced during the treatment period with the FGFR1 inhibitor and can be replaced with a medium supplemented with the FGFR1 inhibitor, having the same composition or a different composition than before the replacement, according to the culture schedule.

[00236] In the present invention, the proportion of Ki67 positive cells in the cells to be treated with the FGFR1 inhibitor (starting material cells) is not particularly limited and may be 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.

[00237] In the present invention, the proportion of alkaline phosphatase positive cells in the cells to be treated with the FGFR1 inhibitor (starting material cells) is not particularly limited and may be

63/74 10% or less, 5% or less, 1% or less, or 0.5% or less.

[00238] The population of endocrine progenitor cells or the population of cells at a later stage of differentiation may be subjected to the additional differentiation stage in the population of cells of interest (for example, a population of insulin-producing cells or a population of cells pancreatic β), in addition to being treated with the FGFR1 inhibitor. As used herein, “in addition to being treated with the FGFR1 inhibitor”; includes the case of performing the treatment step with the FGFR1 inhibitor and the differentiation step at the same time, the case of treating the cell population with the FGFR1 inhibitor, followed by the differentiation step, and the case of submitting the population of cells to the differentiation step, followed by the treatment step with the FGFR1 inhibitor. Thus, the medium for use in the treatment with the FGFR1 inhibitor and the medium for use in differentiating the cell population can be separate media or the medium for use in the differentiation step can be additionally supplemented with the FGFR1 inhibitor.

[00239] In one embodiment, the FGFR1 inhibitor may be contained in a medium for use in the differentiation stage of endocrine progenitor cells into insulin-producing cells and may more preferably be contained in a medium for use in the additional differentiation stage (for example, example, for about the last 4 to 15 days, preferably about the last 4 to 7 days, from step 6) of a population of early insulin-producing cells obtained by inducing the differentiation of a population of endocrine progenitor cells or cells in a stage differentiation. In the case of treating a population of endocrine progenitor cells with the FGFR1 inhibitor, the number of cells of interest can be drastically reduced. In the case of treating a population of an early insulin-producing cell or a cell in a later stage of differentiation with the FGFR1 inhibitor, such

64/74 drastic decrease in the number of cells of interest can be circumvented. Specifically, in the case of treating a population of an early insulin-producing cell or a cell in a later stage of differentiation with the FGFR1 inhibitor, the resulting number of insulin-producing cells can be increased compared to the case of treating insulin. a population of endocrine progenitor cells with the FGFR1 inhibitor.

[00240] Treatment of a population of endocrine progenitor cells or a population of cells at a later stage of differentiation with the FGFR1 inhibitor can inhibit the proliferation of Ki67 positive cells in the cell population.

[00241] This approach can decrease or suppress the number of remaining proliferative cells contained in the pancreatic lineage, not inhibiting the proliferation of teratoma.

[00242] This approach can decrease or suppress the number of remaining proliferative cells contained in the pancreatic lineage, not inhibiting the proliferation of iPS cells (for example, not having to decrease the number of alkaline phosphatase positive cells).

[00243] This approach is able to reduce the absolute number of Ki67 positive cells in a population of endocrine progenitor cells or in a population of cells in a later stage of treatment differentiation using an FGFR1 inhibitor. This can deplete Ki67 positive cells in the cell population obtained.

[00244] Specifically, the proportion of Ki67 positive cells in the cell population obtained can be reduced compared to the case of culture and / or differentiation without treatment with the FGFR1 inhibitor. The proportion can be less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% and can be preferably less than 3%,

65/74 less than 2% or less than 1%.

[00245] According to this approach, the population of endocrine progenitor cells or the population of cells in a later stage of differentiation treated with the FGFR1 inhibitor can be differentiated into insulin-producing cells or pancreatic β cells to obtain a population of cells with inhibited growth of Ki67 positive cells and including insulin-producing cells or enriched pancreatic β cells. Specifically, the proportion of insulin-producing cells or pancreatic β cells in the cell population obtained after induction of differentiation can be increased compared to a cell population obtained without treatment with the FGFR1 inhibitor. The proportion can be 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.

[00246] Insulin-producing cells or pancreatic β cells obtained by this approach can be resided as they are and used as insulin-secreting cells when transplanted into the living body of an animal and differentiated into the living body of the animal. Insulin-producing cells or pancreatic β cells obtained by this approach can bypass the proliferation of Ki67-positive cells and achieve a safe and long-term survival of the transplanted cell graft.

[00247] In accordance with the present invention, the population of insulin-producing cells or the population of pancreatic β cells from which highly proliferative Ki67 positive cells have been removed (cell population of the present invention) is transplanted as is or in the form of capsule to an affected area and is thus useful as a cellular medicine for the treatment of diabetes mellitus, particularly type I diabetes mellitus.

[00248] The cell population of the present invention can be a prodrug. The prodrug refers to a population of cells that is

66/74 differentiated after transplantation into a living body and converted into cells with the function of treating a disease.

[00249] The cell population of the present invention has low toxicity (for example, acute toxicity, chronic toxicity, genetic toxicity, reproductive toxicity, cardiotoxicity and carcinogenicity) and can be safely administered as is or in the form of a pharmaceutical composition containing the cell population mixed with a pharmacologically acceptable carrier, etc. for a mammal (for example, a mouse, a rat, a hamster, a rabbit, a cat, a dog, cattle, sheep, a monkey and a human).

[00250] In the following, the present invention will be described with reference to the Examples. However, the present invention is not limited by these examples. EXAMPLES

[00251] The induction of differentiation of pluripotent stem cells in a population of endocrine progenitor cells was carried out according to steps 1) to 5) above, the previous report (Stem Cell Research (2015) 14, 185-197), etc. Example 1: Decrease in the number of unintended cells (Ki67 positive cells) in the cell population obtained by treating the endocrine progenitor cell population with receptor 1 inhibitor

FGF

1. Method (1) Preparation of the population of insulin-producing cells

[00252] The induction of the differentiation of the endocrine progenitor cell population into an insulin-producing cell population was carried out according to step 6) above or the previous report (Nature Biotechnology 2014; 32: 1121-1133).

[00253] The population of endocrine progenitor cells obtained by

67/74 iPS cell differentiation induction was cultured for 12 days in a differentiation induction medium (improved MEM / 1% B27 / Streptomycin penicillin) containing differentiating factors (ALK5 II, T3, LDN inhibitor, γ- inhibitor secretase RO and ascorbic acid) and an FGF receptor 1 inhibitor (CAS192705-79-6) to obtain an insulin-producing cell population. The number of Ki67 positive cells in the cell population obtained was evaluated by flow cytometry. (2) Transplantation of cells treated with FGF receptor 1 inhibitor in living body

[00254] In the description below, the preparation of mice and the transplantation of cells were performed according to known methods.

[00255] The population of insulin-producing cells obtained by culture in the medium for differentiation induction containing CAS192705-79-6 in the previous section (1) or a population of insulin-producing cells obtained by culture in a medium for induction of free differentiation CAS192705- 79-6 as a comparative example was transplanted under the kidney capsule using immunodeficient NOD / SCID mice with streptozotocin-induced diabetes mellitus. For follow-up after transplantation, human C-peptide concentrations in the blood and blood glucose levels were measured. The grafts were excised 5 weeks after transplantation. (3) Evaluation of unintended cells in the graft

[00256] The excised grafts were fixed and dehydrated, and then the frozen sections were prepared and subjected to immunohistological staining to evaluate the unwanted cells. Cells of interest that could mature in pancreatic islet cells in vivo were assessed using either insulin positivity (and NKX6.1 positivity) or glucagon positivity as an indicator, and unintended cells with the possibility of adversely affecting graft survival The

68/74 long term cells of interest were evaluated using Ki67 positivity as an indicator.

2. Results (1) Preparation of the population of insulin-producing cells

[00257] An experiment to treat cells for 12 days with a differentiation-inducing medium containing CAS192705-79-6 (1 µM) was performed five times. The results on the proportion of standard deviation of cells positive for Ki67 ± in the population of cells obtained are shown in Table 1. In the case of treatment of a population of endocrine progenitor cells with CAS192705-79-6 for 12 days, the proportion of cells positive for Ki67 in the cell population obtained was much lower than in a control. These results indicate that the treatment of a population of endocrine progenitor cells with CAS192705-79-6 decreases the number of Ki67 positive cells or inhibits their proliferation. [Table 1] Absent FGF receptor inhibitor treatment (1 µM) Percentage of Ki67 positive cells 10.78 ± 4.09% 0.98 ± 0.29% (before transplantation) (2) Transplantation of cells treated with FGF receptor 1 inhibitor in living body

[00258] Results on human C-peptide concentrations in blood and blood glucose levels measured for follow-up after transplantation are shown in Table 2. The population of insulin-producing cells obtained by treatment for 12 days with a medium for differentiation induction containing CAS192705-79-6 (1 µM) secreted human C peptide in the blood and showed an elevated blood glucose-enhancing effect, 4 months after transplantation. Their levels were equivalent to those in the case of transplantation of a population of insulin-producing cells obtained without treatment with CAS192705-79-6. [Table 2]

69/74 Treatment with FGF receptor inhibitor absent present (1 µM) Human C-peptide concentration in the blood 4 months after transplant 1440 ± 573 1486 ± 305 (pM) Blood glucose level at the time of 516 ± 35 (at moment of 516 ± 33 (at the time of transplant and 4 months after transplant transplant) transplant) (mg / dL) 85 ± 16 (4 months later) 86 ± 22 (4 months later) (3) Evaluation of unintended cells in the graft

[00259] The results of the immunohistological staining grafts excised 5 weeks after the transplant are shown in Figure 1.

[00260] In the case of transplantation, under the kidney capsule, a population of insulin-producing cells obtained by treatment for 12 days with a differentiation-inducing medium containing CAS192705-79-6 (1 µM), drastic inhibition of the swelling of the Grafts were verified in comparison with the transplant case, under the kidney capsule, a population of insulin-producing cells obtained without treatment with CAS192705-79-6. These results indicate that treating a population of endocrine progenitor cells with CAS192705-79-6 decreases the number of Ki67 positive cells, which are unintentional cells, or inhibits their proliferation. Example 2: Decrease in the number of unwanted cells (Ki67 positive cells) and maintenance of cells of interest (insulin positive cells (and NKX6.1 positive cells)) in the cell population obtained by treating the population of insulin-producing cells with FGF receptor 1 inhibitor

1. Method

[00261] 1) A population of endocrine progenitor cells obtained by inducing differentiation of iPS cells was cultured for 11 days in a medium for inducing differentiation (improved MEM / 1% B27 / Streptomycin penicillin) containing differentiating factors (ALK5 inhibitor II, T3, LDN, inhibitor of γ-secretase RO and ascorbic acid) and an inhibitor of FGF receptor 1 (CAS192705-79-6) to obtain a population of insulin-producing cells.

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[00262] 2) A population of endocrine progenitor cells obtained by inducing differentiation of iPS cells was cultured for 4 days in a medium for induction of differentiation (improved MEM / 1% B27 / Streptomycin penicillin) containing differentiating factors (ALK5 inhibitor II, T3, LDN, γ-secretase RO inhibitor and ascorbic acid) and thus induced to differentiate in a population of insulin-producing cells. Subsequently, an FGF receptor 1 inhibitor (CAS192705-79-6, 1 µM) was added to a differentiation induction medium (improved MEM / 1% B27 / streptomycin penicillin) containing differentiating factors (ALK5 II, T3 inhibitor , LDN, RO γ-secretase inhibitor and ascorbic acid), and the cells were grown there for 7 days.

[00263] 3) A population of endocrine progenitor cells obtained by inducing differentiation of iPS cells was cultured for 7 days in a medium for differentiation induction (improved MEM / 1% B27 / Streptomycin penicillin) containing differentiating factors (ALK5 inhibitor II, T3, LDN, γ-secretase RO inhibitor and ascorbic acid) and thus induced to differentiate in a population of insulin-producing cells. Subsequently, an FGF receptor 1 inhibitor (CAS192705-79-6, 1 µM) was added to a differentiation induction medium (improved MEM / 1% B27 / streptomycin penicillin) containing differentiating factors (ALK5 II, T3 inhibitor , LDN, γ-secretase RO inhibitor and ascorbic acid), and the cells were grown there for 4 days.

[00264] 4) A population of endocrine progenitor cells obtained by inducing differentiation of iPS cells was cultured for 11 days in a medium for inducing differentiation (improved MEM / 1% B27 / Streptomycin penicillin) containing differentiating factors (ALK5 inhibitor II, T3, LDN, RO γ-secretase inhibitor and ascorbic acid) and not containing CAS192705-79-6 and thus induced to differentiate in an insulin-producing cell population.

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[00265] The number of Ki67 positive cells in the cell population obtained by each of methods 1), 2) and 3) above was counted by flow cytometry to determine the proportion of Ki67 positive cells in each cell population.

[00266] The number of cells positive for insulin (and positive for NKX6.1) in the cell population obtained by each of methods 1), 2) and 3) above was counted by flow cytometry. The proportion of cells positive for insulin and (positive for NKX6.1) in each method was calculated as a relative value when the number of positive cells for insulin (and positive for NKX6.1) in the control cell population obtained by method 4 ) above was defined as 100%.

2. Results

[00267] An experiment using each method was repeated three times to assess the influence of the timing of treatment with CAS192705-79-6 on an insulin-producing cell production stage. The results on the average proportions of cells positive for Ki67 and positive cells for insulin (and positive for NKX6.1) obtained in each ± standard deviation of the method are shown in Table 3.

[00268] The proportion of Ki67 positive cells in the cell population obtained did not differ significantly between the case of cell treatment using CAS192705-79-6 from the beginning of the differentiation induction step, the case of cell treatment in the last 7 days, and the case of cell treatment in the last 4 days, being much lower in all cases than that of the control. These results indicate that treatment with CAS192705-79-6 in the production process of insulin-producing cells or pancreatic β cells can decrease the number of Ki67 positive cells in the cell population or inhibit their proliferation.

[00269] On the other hand, the proportion of cells positive for insulin (and positive for NKX6.1) differed markedly between the case of treatment

72/74 cells since the beginning of the differentiation induction stage and the case of cell treatment in the last 7 days or 4 days. Specifically, in the case of treating cells (population of endocrine progenitor cells) with CAS192705-79-6 since the start of the differentiation induction stage, it was confirmed that the number of cells positive for insulin (and positive for NKX6.1), which were the cells of interest, declined sharply. In contrast, in the case of treatment of cells (population of early insulin-producing cells or cells at a later stage of differentiation) with CAS192705-79-6 at a stage where differentiation has progressed to some extent 4 days or 7 days after beginning of differentiation induction, it was confirmed that the number of insulin-positive (and NKX6.1-positive) cells, which were the cells of interest, rarely decreased. [Table 3] Present (1 µM) Present (1 µM) Treatment with inhibitor of Present (1 µM) Absent In the last 7 In the last 4 FGF receptor From the beginning days days Cell proportion 7.57 ± 1.05% 0 , 57 ± 0.35% 0.47 ± 0.15% 0.57 ± 0.15% positive for Ki67 Proportion of cells positive for insulin and 100 ± 18% 45 ± 15% 88 ± 13% 91 ± 16% ( positive for NKX6.1) Example 3: Decrease in the number of unintended cells (Ki67 positive cells) in the cell population obtained by treatment with FGFR1 inhibitor other than CAS192705-79-6

1. Method

[00270] A population of endocrine progenitor cells obtained by inducing differentiation of iPS cells was cultured for 8 days in a medium for inducing differentiation (improved MEM / 1% B27 / Streptomycin penicillin) containing differentiating factors (ALK5 II inhibitor, T3, LDN, RO γ-secretase inhibitor and ascorbic acid). Subsequently, CAS192705-79-6, E-3810 or PD173074 was added in three concentrations (low, medium and high) to a medium for differentiation induction (MCDB131 / 20 mM glucose / NaHCO3 / FAF-BSA / ITS-X / Glutamax /acid

73/74 ascorbic / Penicillin Streptomycin) containing differentiating factors (T3, ALK5 II inhibitor, ZnSO4, heparin, N-acetylcysteine, Trolox and R428), and the cells were cultured in the medium for 4 days. The number of Ki67 positive cells in the cell population obtained was evaluated by flow cytometry.

2. Results

[00271] The proportion of Ki67 positive cells in a cell population obtained by treatment with CAS192705-79-6 or E-3810 is shown in Table 4. In addition, the proportion of Ki67 positive cells in a cell population obtained by treatment with PD173074 is shown in Table 5.

[00272] From these results, it was confirmed that in cell populations obtained by treatment not only with CAS192705-79-6, but with other FGFR1 inhibitors, the proportion of Ki67 positive cells was much lower than that of the control. These results indicate that the treatment of FGFR1 inhibition without being limited by treatment with CAS192705-79- 6 decreases the number of Ki67 positive cells or inhibits their proliferation. [Table 4] Proportion of Ki67 positive cells (%); FGFR1 inhibitor Treatment concentration (µM) Absent 4.13% - - 2.15%; 0.99%; 0.7%; CAS192705-79-6 0.1 µM 0.3 µM 1 µM 2.74%; 1.51%; 0.67%; E-3810 0.3 µM 1 µM 10 µM [Table 5] Proportion of Ki67-positive cells (%); FGFR1 inhibitor Treatment concentration (µM) Absent 2.7% - - 0.7%; 0.7%; 0.5%; PD-173074 0.1 µM 1 µM 5 µM

[00273] The results described above demonstrated that treatment of a population of endocrine progenitor cells or cells in a later stage of differentiation with an FGFR1 inhibitor is able to decrease the number of Ki67 positive cells present in the population of

74/74 cells or inhibit their proliferation, and as a result, a population of cells enriched in the cells of interest can be obtained.

Claims (14)

1. Method for producing a population of insulin-producing cells or a population of pancreatic β cells, characterized by the fact that it comprises the step of treating a population of insulin-producing cells or a population of pancreatic β cells with an FGFR1 inhibitor . 2. Method according to claim 1, characterized by the fact that it additionally comprises the stage of differentiation of the population of insulin-producing cells treated with the FGFR1 inhibitor. Method according to claim 1 or 2, characterized by the fact that the population of insulin-producing cells or the population of pancreatic β cells is treated with less than 5 µM of the FGFR1 inhibitor. Method according to any one of claims 1 to 3, characterized in that the cell population produced comprises cells positive for Ki67 in a proportion of less than 3%. Method according to any one of claims 1 to 4, characterized in that the FGFR1 inhibitor is 1- [2-amino-6- (3,5-dimethoxyphenyl) -pyrido (2,3-d) pyrimidin -7-yl] -3-tert-butylurea or a salt thereof. Method according to any one of claims 2 to 5, characterized in that the step of differentiating the population of insulin-producing cells treated with the FGFR1 inhibitor is carried out by transplantation to an animal. 7. Method to produce a population of insulin-producing cells or a population of pancreatic β cells, characterized by the fact that it comprises the stage of treatment of a population of endocrine progenitor cells or cells in a later stage of differentiation with less than 5 µM of an FGFR1 inhibitor. 8. Method to decrease the number or inhibit the proliferation of Ki67 positive cells present in a population of endocrine progenitor cells or cells in a later stage of differentiation, characterized by the fact that it comprises treating the cell population with an FGFR1 inhibitor. Method according to claim 8, characterized in that the population of endocrine progenitor cells or cells in a later stage of differentiation is a population of insulin-producing cells. Method according to claim 8 or 9, characterized in that the population of endocrine progenitor cells or cells in a later stage of differentiation is treated with less than 5 µM of the FGFR1 inhibitor. Method according to any one of claims 8 to 10, characterized in that the FGFR1 inhibitor is 1- [2-amino-6- (3,5-dimethoxyphenyl) -pyrido (2,3-d) pyrimidin -7-yl] -3-tert-butylurea or a salt thereof. 12. Population of pancreatic progenitor cells or cells in a later stage of differentiation, characterized by the fact that it comprises Ki67 positive cells in a proportion of less than 3%. 13. Cell population according to claim 12, characterized by the fact that the cell population is a population of insulin-producing cells. 14. Cell population according to claim 12 or 13, characterized by the fact that the cell population is used for transplantation.